Mutant receptors and their use in a nuclear receptor-based inducible gene expression system

ABSTRACT

This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates to novel substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system and methods of modulating the expression of a gene in a host cell for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic organisms.

This application claims priority to U.S. provisional application No.60/567,294 filed Apr. 30, 2004 and U.S. provisional application No.60/609,424 filed Sep. 13, 2004.

FIELD OF THE INVENTION

This invention relates to the field of biotechnology or geneticengineering. Specifically, this invention relates to the field of geneexpression. More specifically, this invention relates to novel nuclearreceptors comprising a substitution mutation and their use in a nuclearreceptor-based inducible gene expression system and methods ofmodulating the expression of a gene within a host cell using thisinducible gene expression system.

BACKGROUND OF THE INVENTION

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties. However, the citation ofany reference herein should not be construed as an admission that suchreference is available as “Prior Art” to the instant application.

In the field of genetic engineering, precise control of gene expressionis a valuable tool for studying, manipulating, and controllingdevelopment and other physiological processes. Gene expression is acomplex biological process involving a number of specificprotein-protein interactions. In order for gene expression to betriggered, such that it produces the RNA necessary as the first step inprotein synthesis, a transcriptional activator must be brought intoproximity of a promoter that controls gene transcription. Typically, thetranscriptional activator itself is associated with a protein that hasat least one DNA binding domain that binds to DNA binding sites presentin the promoter regions of genes. Thus, for gene expression to occur, aprotein comprising a DNA binding domain and a transactivation domainlocated at an appropriate distance from the DNA binding domain must bebrought into the correct position in the promoter region of the gene.

The traditional transgenic approach utilizes a cell-type specificpromoter to drive the expression of a designed transgene. A DNAconstruct containing the transgene is first incorporated into a hostgenome. When triggered by a transcriptional activator, expression of thetransgene occurs in a given cell type.

Another means to regulate expression of foreign genes in cells isthrough inducible promoters. Examples of the use of such induciblepromoters include the PR1-a promoter, prokaryotic repressor-operatorsystems, immunosuppressive-immunophilin systems, and higher eukaryotictranscription activation systems such as ecdysteroid hormone receptorsystems and are described below.

The PR1-a promoter from tobacco is induced during the systemic acquiredresistance response following pathogen attack. The use of PR1-a may belimited because it often responds to endogenous materials and externalfactors such as pathogens, UV-B radiation, and pollutants. Generegulation systems based on promoters induced by heat shock, interferonand heavy metals have been described (Wurn et al., 1986, Proc. Natl.Acad. Sci. USA 83: 5414-5418; Arnheiter et al., 1990, Cell 62:51-61;Filmus et al., 1992, Nucleic Acids Research 20: 27550-27560). However,these systems have limitations due to their effect on expression ofnon-target genes. These systems are also leaky.

Prokaryotic repressor-operator systems utilize bacterial repressorproteins and the unique operator DNA sequences to which they bind. Boththe tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systemsfrom the bacterium Escherichia coli have been used in plants and animalsto control gene expression. In the Tet system, tetracycline binds to theTetR repressor protein, resulting in a conformational change thatreleases the repressor protein from the operator which as a resultallows transcription to occur. In the Lac system, a lac operon isactivated in response to the presence of lactose, or synthetic analogssuch as isopropyl-b-D-thiogalactoside. Unfortunately, the use of suchsystems is restricted by unstable chemistry of the ligands, i.e.tetracycline and lactose, their toxicity, their natural presence, or therelatively high levels required for induction or repression. For similarreasons, utility of such systems in animals is limited.

Immunosuppressive molecules such as FK506, rapamycin and cyclosporine Acan bind to immunophilins FKBP12, cyclophilin, etc. Using thisinformation, a general strategy has been devised to bring together anytwo proteins simply by placing FK506 on each of the two proteins or byplacing FK506 on one and cyclosporine A on another one. A synthetichomodimer of FK506 (FK1012) or a compound resulted from fusion ofFK506-cyclosporine (FKCsA) can then be used to induce dimerization ofthese molecules (Spencer et al., 1993, Science 262: 1019-24; Belshaw etal., 1996 Proc Natl Acad Sci USA 93: 4604-7). Gal4 DNA binding domainfused to FKBP12 and VP16 activator domain fused to cyclophilin, andFKCsA compound were used to show heterodimerization and activation of areporter gene under the control of a promoter containing Gal4 bindingsites. Unfortunately, this system includes immunosuppressants that canhave unwanted side effects and therefore, limits its use for variousmammalian gene switch applications.

Higher eukaryotic transcription activation systems such as steroidhormone receptor systems have also been employed. Steroid hormonereceptors are members of the nuclear receptor superfamily and are foundin vertebrate and invertebrate cells. Unfortunately, use of steroidalcompounds that activate the receptors for the regulation of geneexpression, particularly in plants and mammals, is limited due to theirinvolvement in many other natural biological pathways in such organisms.In order to overcome such difficulties, an alternative system has beendeveloped using insect ecdysone receptors (EcR).

Growth, molting, and development in insects are regulated by theecdysone steroid hormone (molting hormone) and the juvenile hormones(Dhadialla, et al., 1998, Annu. Rev. Entomol. 43: 545-569). Themolecular target for ecdysone in insects consists of at least ecdysonereceptor (EcR) and ultraspiracle protein (USP). EcR is a member of thenuclear steroid receptor super family that is characterized by signatureDNA and ligand binding domains, and an activation domain (Koelle et al.1991, Cell, 67:59-77). EcR receptors are responsive to a number ofecdysteroidal compounds such as ponasterone A and muristerone A.Recently, non-steroidal compounds with ecdysteroid agonist activity havebeen described, including the commercially available insecticidestebufenozide and methoxyfenozide that are marketed world wide by Rohmand Haas Company (see International Patent Application No.PCT/EP96/00686 and U.S. Pat. No. 5,530,028). Both analogs haveexceptional safety profiles to other organisms.

The insect ecdysone receptor (EcR) heterodimerizes with Ultraspiracle(USP), the insect homologue of the mammalian RXR, and binds ecdysteroidsand ecdysone receptor response elements and activate transcription ofecdysone responsive genes (Riddiford et al., 2000). The EcR/USP/ligandcomplexes play important roles during insect development andreproduction. The EcR is a member of the steroid hormone receptorsuperfamily and has five modular domains, A/B (transactivation), C (DNAbinding, heterodimerization)), D (Hinge, heterodimerization), E (ligandbinding, heterodimerization and transactivation and F (transactivation)domains. Some of these domains such as A/B, C and E retain theirfunction when they are fused to other proteins.

Tightly regulated inducible gene expression systems or “gene switches”are useful for various applications such as gene therapy, large scaleproduction of proteins in cells, cell based high throughput screeningassays, functional genomics and regulation of traits in transgenicplants and animals.

The first version of EcR-based gene switch used Drosophila melanogasterEcR (DmEcR) and Mus musculus RXR (MmRXR) and showed that these receptorsin the presence of ecdysteroid, ponasteroneA, transactivate reportergenes in mammalian cell lines and transgenic mice (Christopherson etal., 1992; No et al., 1996). Later, Suhr et al., 1998 showed thatnon-ecdysteroidal ecdysone agonist, tebufenozide, induced high level oftransactivation of reporter genes in mammalian cells through Bombyx moriEcR (BrnEcR) in the absence of exogenous heterodimer partner.

International Patent Applications No. PCT/US97/05330 (WO 97/38117) andPCT/US99/08381 (WO 99/58155) disclose methods for modulating theexpression of an exogenous gene in which a DNA construct comprising theexogenous gene and an ecdysone response element is activated by a secondDNA construct comprising an ecdysone receptor that, in the presence of aligand therefor, and optionally in the presence of a receptor capable ofacting as a silent partner, binds to the ecdysone response element toinduce gene expression. The ecdysone receptor of choice was isolatedfrom Drosophila melanogaster. Typically, such systems require thepresence of the silent partner, preferably retinoid X receptor (RXR), inorder to provide optimum activation. In mammalian cells, insect ecdysonereceptor (EcR) heterodimerizes with retinoid X receptor (RXR) andregulates expression of target genes in a ligand dependent manner.International Patent Application No. PCT/US98/14215 (WO 99/02683)discloses that the ecdysone receptor isolated from the silk moth Bombyxmori is functional in mammalian systems without the need for anexogenous dimer partner.

U.S. Pat. No. 6,265,173 B1 discloses that various members of thesteroid/thyroid superfamily of receptors can combine with Drosophilamelanogaster ultraspiracle receptor (USP) or fragments thereofcomprising at least the dimerization domain of USP for use in a geneexpression system. U.S. Pat. No. 5,880,333 discloses a Drosophilamelanogaster EcR and ultraspiracle (USP) heterodimer system used inplants in which the transactivation domain and the DNA binding domainare positioned on two different hybrid proteins. Unfortunately, theseUSP-based systems are constitutive in animal cells and therefore, arenot effective for regulating reporter gene expression.

In each of these cases, the transactivation domain and the DNA bindingdomain (either as native EcR as in International Patent Application No.PCT/US98/14215 or as modified EcR as in International Patent ApplicationNo. PCT/US97/05330) were incorporated into a single molecule and theother heterodimeric partners, either USP or RXR, were used in theirnative state.

Drawbacks of the above described EcR-based gene regulation systemsinclude a considerable background activity in the absence of ligands andnon-applicability of these systems for use in both plants and animals(see U.S. Pat. No. 5,880,333). Therefore, a need exists in the art forimproved EcR-based systems to precisely modulate the expression ofexogenous genes in both plants and animals. Such improved systems wouldbe useful for applications such as gene therapy, large-scale productionof proteins and antibodies, cell-based high throughput screening assays,functional genomics and regulation of traits in transgenic animals. Forcertain applications such as gene therapy, it may be desirable to havean inducible gene expression system that responds well to syntheticnon-ecdysteroid ligands and at the same is insensitive to the naturalecdysteroids. Thus, improved systems that are simple, compact, anddependent on ligands that are relatively inexpensive, readily availableand of low toxicity to the host would prove useful for regulatingbiological systems.

Previously, Applicants have shown that an ecdysone receptor-basedinducible gene expression system in which the transactivation and DNAbinding domains are separated from each other by placing them on twodifferent proteins results in greatly reduced background activity in theabsence of a ligand and significantly increased activity over backgroundin the presence of a ligand (pending application PCT/US01/09050,incorporated herein in its entirety by reference). This two-hybridsystem is a significantly improved inducible gene expression modulationsystem compared to the two systems disclosed in applicationsPCT/US97/05330 and PCT/US98/14215. The two-hybrid system exploits theability of a pair of interacting proteins to bring the transcriptionactivation domain into a more favorable position relative to the DNAbinding domain such that when the DNA binding domain binds to the DNAbinding site on the gene, the transactivation domain more effectivelyactivates the promoter (see, for example, U.S. Pat. No. 5,283,173).Briefly, the two-hybrid gene expression system comprises two geneexpression cassettes; the first encoding a DNA binding domain fused to anuclear receptor polypeptide, and the second encoding a transactivationdomain fused to a different nuclear receptor polypeptide. In thepresence of ligand, the interaction of the first polypeptide with thesecond polypeptide effectively tethers the DNA binding domain to thetransactivation domain. Since the DNA binding and transactivationdomains reside on two different molecules, the background activity inthe absence of ligand is greatly reduced.

A two-hybrid system also provides improved sensitivity tonon-ecdysteroidal ligands for example, diacylhydrazines, when comparedto ecdysteroidal ligands for example, ponasterone A (“PonA”) ormuristerone A (“MurA”). That is, when compared to ecdysteroids, thenon-ecdysteroidal ligands provide higher activity at a lowerconcentration. In addition, since transactivation based on EcR geneswitches is often cell-line dependent, it is easier to tailor switchingsystems to obtain maximum transactivation capability for eachapplication. Furthermore, the two-hybrid system avoids some side effectsdue to overexpression of RXR that often occur when unmodified RXR isused as a switching partner. In a preferred two-hybrid system, nativeDNA binding and transactivation domains of EcR or RXR are eliminated andas a result, these hybrid molecules have less chance of interacting withother steroid hormone receptors present in the cell resulting in reducedside effects.

The EcR is a member of the nuclear receptor superfamily and classifiedinto subfamily 1, group H (referred to herein as “Group H nuclearreceptors”). The members of each group share 40-60% amino acid identityin the E (ligand binding) domain (Laudet et al., A Unified NomenclatureSystem for the Nuclear Receptor Subfamily, 1999; Cell 97: 161-163). Inaddition to the ecdysone receptor, other members of this nuclearreceptor subfamily 1, group H include: ubiquitous receptor (UR), Orphanreceptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1), RXRinteracting protein-15 (RIP-15), liver x receptor β (LXRβ), steroidhormone receptor like protein (RLD-1), liver x receptor (LXR), liver xreceptor α (LXRα), farnesoid x receptor (FXR), receptor interactingprotein 14 (RIP-14), and farnesol receptor (HRR-1).

To develop an improved Group H nuclear receptor-based inducible geneexpression system in which ligand binding or ligand specificity ismodified, Applicants created substitution mutant EcRs that comprisesubstituted amino acid residues in the ligand binding domain (LBD). Ahomology modeling and docking approach was used to predict criticalresidues that mediate binding of ecdysteroids and non-ecdysteroids tothe EcR LBD. These substitution mutant EcRs were evaluated in ligandbinding and transactivation assays. As presented herein, Applicants'novel substitution mutant nuclear receptors and their use in a nuclearreceptor-based inducible gene expression system provides an improvedinducible gene expression system in both prokaryotic and eukaryotic hostcells in which ligand sensitivity and magnitude of transactivation maybe selected as desired, depending upon the application.

DETAILED DESCRIPTION OF THE INVENTION

Applicants describe herein the construction of Group H nuclear receptorsthat comprise substitution mutations (referred to herein as“substitution mutants”) at amino acid residues that are involved inligand binding to a Group H nuclear receptor ligand binding domain thataffect the ligand sensitivity and magnitude of induction of the Group Hnuclear receptor and the demonstration that these substitution mutantnuclear receptors are useful in methods of modulating gene expression.

Specifically, Applicants have developed a novel nuclear receptor-basedinducible gene expression system comprising a Group H nuclear receptorligand binding domain comprising a substitution mutation. Applicantshave shown that the effect of such a substitution mutation may increaseor reduce ligand binding activity or ligand sensitivity and the ligandmay be ecdysteroid or non-ecdysteroid specific. Thus, Applicants'invention provides a Group H nuclear receptor-based inducible geneexpression system useful for modulating expression of a gene of interestin a host cell. In a particularly desirable embodiment, Applicants'invention provides an ecdysone receptor-based inducible gene expressionsystem that responds solely to either ecdysteroidal ligands ornon-ecdysteroidal ligands. In addition, the present invention alsoprovides an improved non-ecdysteroidal ligand responsive ecdysonereceptor-based inducible gene expression system. Thus, Applicants' novelinducible gene expression system and its use in methods of modulatinggene expression in a host cell overcome the limitations of currentlyavailable inducible expression systems and provide the skilled artisanwith an effective means to control gene expression.

The present invention is useful for applications such as gene therapy,large scale production of proteins and antibodies, cell-based highthroughput screening assays, orthogonal ligand screening assays,functional genomics, proteomics, metabolomics, and regulation of traitsin transgenic organisms, where control of gene expression levels isdesirable. An advantage of Applicants' invention is that it provides ameans to regulate gene expression and to tailor expression levels tosuit the user's requirements.

DEFINITIONS

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided and should be helpful inunderstanding the scope and practice of the present invention.

In a specific embodiment, the term “about” or “approximately” meanswithin 20%, preferably within 10%, more preferably within 5%, and evenmore preferably within 1% of a given value or range.

The term “substantially free” means that a composition comprising “A”(where “A” is a single protein, DNA molecule, vector, recombinant hostcell, etc.) is substantially free of “B” (where “B” comprises one ormore contaminating proteins, DNA molecules, vectors, etc.) when at leastabout 75% by weight of the proteins, DNA, vectors (depending on thecategory of species to which A and B belong) in the composition is “A”.Preferably, “A” comprises at least about 90% by weight of the A+Bspecies in the composition, most preferably at least about 99% byweight. It is also preferred that a composition, which is substantiallyfree of contamination, contain only a single molecular weight specieshaving the activity or characteristic of the species of interest.

The term “isolated” for the purposes of the present invention designatesa biological material (nucleic acid or protein) that has been removedfrom its original environment (the environment in which it is naturallypresent). For example, a polynucleotide present in the natural state ina plant or an animal is not isolated, however the same polynucleotideseparated from the adjacent nucleic acids in which it is naturallypresent, is considered “isolated”. The term “purified” does not requirethe material to be present in a form exhibiting absolute purity,exclusive of the presence of other compounds. It is rather a relativedefinition.

A polynucleotide is in the “purified” state after purification of thestarting material or of the natural material by at least one order ofmagnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

A “nucleic acid” is a polymeric compound comprised of covalently linkedsubunits called nucleotides. Nucleic acid includes polyribonucleic acid(RNA) and polydeoxyribonucleic acid (DNA), both of which may besingle-stranded or double-stranded. DNA includes but is not limited tocDNA, genomic DNA, plasmids DNA, synthetic DNA, and semi-synthetic DNA.DNA may be linear, circular, or supercoiled.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

The term “fragment” will be understood to mean a nucleotide sequence ofreduced length relative to the reference nucleic acid and comprising,over the common portion, a nucleotide sequence identical to thereference nucleic acid. Such a nucleic acid fragment according to theinvention may be, where appropriate, included in a larger polynucleotideof which it is a constituent. Such fragments comprise, or alternativelyconsist of, oligonucleotides ranging in length from at least 6, 8, 9,10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51,54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200,300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a nucleicacid according to the invention.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “gene” refers to an assembly of nucleotides that encode a polypeptide,and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to anucleic acid fragment that expresses a specific protein or polypeptide,including regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. “Native gene”refers to a gene as found in nature with its own regulatory sequences.“Chimeric gene” refers to any gene that is not a native gene, comprisingregulatory and/or coding sequences that are not found together innature. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A chimericgene may comprise coding sequences derived from different sources and/orregulatory sequences derived from different sources. “Endogenous gene”refers to a native gene in its natural location in the genome of anorganism. A “foreign” gene or “heterologous” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

The term “genome” includes chromosomal as well as mitochondrial,chloroplast and viral DNA or RNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., 1989 infra). Hybridization andwashing conditions are well known and exemplified in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989), particularly Chapter 11 and Table 11.1 therein (entirelyincorporated herein by reference). The conditions of temperature andionic strength determine the “stringency” of the hybridization.

Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC,0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5%SDS). Moderate stringency hybridization conditions correspond to ahigher T_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The term “complementary” is usedto describe the relationship between nucleotide bases that are capableof hybridizing to one another. For example, with respect to DNA,adenosine is complementary to thymine and cytosine is complementary toguanine. Accordingly, the instant invention also includes isolatednucleic acid fragments that are complementary to the complete sequencesas disclosed or used herein as well as those substantially similarnucleic acid sequences.

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step atT_(m) of 55° C., and utilizing conditions as set forth above. In apreferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 63° C.; in an even more preferred embodiment,the T_(m) is 65° C.

Post-hybridization washes also determine stringency conditions. One setof preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 minutes (min), then repeated with2×SSC, 0.5% SDS at 45° C. for 30 minutes, and then repeated twice with0.2×SSC, 0.5% SDS at 50° C. for 30 minutes. A more preferred set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Anotherpreferred set of highly stringent conditions uses two final washes in0.1×SSC, 0.1% SDS at 65° C. Hybridization requires that the two nucleicacids comprise complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.

The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8).

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step inless than 500 mM salt and at least 37 degrees Celsius, and a washingstep in 2×SSPE at least 63 degrees Celsius. In a preferred embodiment,the hybridization conditions comprise less than 200 mM salt and at least37 degrees Celsius for the hybridization step. In a more preferredembodiment, the hybridization conditions comprise 2×SSPE and 63 degreesCelsius for both the hybridization and washing steps.

In one embodiment, the length for a hybridizable nucleic acid is atleast about 10 nucleotides. Preferable a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and most preferably the lengthis at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as length of theprobe.

The term “probe” refers to a single-stranded nucleic acid molecule thatcan base pair with a complementary single stranded target nucleic acidto form a double-stranded molecule.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 18 nucleotides, that is hybridizable to a genomicDNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. A labeled oligonucleotide can be used as a probe to detectthe presence of a nucleic acid. Oligonucleotides (one or both of whichmay be labeled) can be used as PCR primers, either for cloning fulllength or a fragment of a nucleic acid, or to detect the presence of anucleic acid. An oligonucleotide can also be used to form a triple helixwith a DNA molecule. Generally, oligonucleotides are preparedsynthetically, preferably on a nucleic acid synthesizer. Accordingly,oligonucleotides can be prepared with non-naturally occurringphosphoester analog bonds, such as thioester bonds, etc.

A “primer” is an oligonucleotide that hybridizes to a target nucleicacid sequence to create a double stranded nucleic acid region that canserve as an initiation point for DNA synthesis under suitableconditions. Such primers may be used in a polymerase chain reaction.

“Polymerase chain reaction” is abbreviated PCR and means an in vitromethod for enzymatically amplifying specific nucleic acid sequences. PCRinvolves a repetitive series of temperature cycles with each cyclecomprising three stages: denaturation of the template nucleic acid toseparate the strands of the target molecule, annealing a single strandedPCR oligonucleotide primer to the template nucleic acid, and extensionof the annealed primer(s) by DNA polymerase. PCR provides a means todetect the presence of the target molecule and, under quantitative orsemi-quantitative conditions, to determine the relative amount of thattarget molecule within the starting pool of nucleic acids.

“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCRand means an in vitro method for enzymatically producing a target cDNAmolecule or molecules from an RNA molecule or molecules, followed byenzymatic amplification of a specific nucleic acid sequence or sequenceswithin the target cDNA molecule or molecules as described above. RT-PCRalso provides a means to detect the presence of the target molecule and,under quantitative or semi-quantitative conditions, to determine therelative amount of that target molecule within the starting pool ofnucleic acids.

A DNA “coding sequence” is a double-stranded DNA sequence that istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.“Suitable regulatory sequences” refer to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and evensynthetic DNA sequences. If the coding sequence is intended forexpression in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding sequence.

“Open reading frame” is abbreviated ORF and means a length of nucleicacid sequence, either DNA, cDNA or RNA, that comprises a translationstart signal or initiation codon, such as an ATG or AUG, and atermination codon and can be potentially translated into a polypeptidesequence.

The term “head-to-head” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-head orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 5′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds away from the 5′ end ofthe other polynucleotide. The term “head-to-head” may be abbreviated(5′)-to-(5′) and may also be indicated by the symbols (←→) or(3′←5′5′→3′).

The term “tail-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a tail-to-tail orientation when the 3′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds toward the otherpolynucleotide. The term “tail-to-tail” may be abbreviated (3′)-to-(3′)and may also be indicated by the symbols (→←) or (5′→3′3′←5′).

The term “head-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-tail orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds in the same directionas that of the other polynucleotide. The term “head-to-tail” may beabbreviated (5′)-to-(3′) and may also be indicated by the symbols (→→)or (5′→3′5′→3′).

The term “downstream” refers to a nucleotide sequence that is located 3′to reference nucleotide sequence. In particular, downstream nucleotidesequences generally relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to reference nucleotide sequence. In particular, upstream nucleotidesequences generally relate to sequences that are located on the 5′ sideof a coding sequence or starting point of transcription. For example,most promoters are located upstream of the start site of transcription.

The terms “restriction endonuclease” and “restriction enzyme” refer toan enzyme that binds and cuts within a specific nucleotide sequencewithin double stranded DNA.

“Homologous recombination” refers to the insertion of a foreign DNAsequence into another DNA molecule, e.g., insertion of a vector in achromosome. Preferably, the vector targets a specific chromosomal sitefor homologous recombination. For specific homologous recombination, thevector will contain sufficiently long regions of homology to sequencesof the chromosome to allow complementary binding and incorporation ofthe vector into the chromosome. Longer regions of homology, and greaterdegrees of sequence similarity, may increase the efficiency ofhomologous recombination.

Several methods known in the art may be used to propagate apolynucleotide according to the invention. Once a suitable host systemand growth conditions are established, recombinant expression vectorscan be propagated and prepared in quantity. As described herein, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

A “vector” is any means for the cloning of and/or transfer of a nucleicacid into a host cell. A vector may be a replicon to which another DNAsegment may be attached so as to bring about the replication of theattached segment. A “replicon” is any genetic element (e.g., plasmid,phage, cosmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo, i.e., capable of replication under its owncontrol. The term “vector” includes both viral and nonviral means forintroducing the nucleic acid into a cell in vitro, ex vivo or in vivo. Alarge number of vectors known in the art may be used to manipulatenucleic acids, incorporate response elements and promoters into genes,etc. Possible vectors include, for example, plasmids or modified virusesincluding, for example bacteriophages such as lambda derivatives, orplasmids such as pBR322 or pUC plasmid derivatives, or the Bluescriptvector. For example, the insertion of the DNA fragments corresponding toresponse elements and promoters into a suitable vector can beaccomplished by ligating the appropriate DNA fragments into a chosenvector that has complementary cohesive termini. Alternatively, the endsof the DNA molecules may be enzymatically modified or any site may beproduced by ligating nucleotide sequences (linkers) into the DNAtermini. Such vectors may be engineered to contain selectable markergenes that provide for the selection of cells that have incorporated themarker into the cellular genome. Such markers allow identificationand/or selection of host cells that incorporate and express the proteinsencoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used include but are notlimited to retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

The term “plasmid” refers to an extra chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction which is capable of introducing apromoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A “cloning vector” is a “replicon”, which is a unit length of a nucleicacid, preferably DNA, that replicates sequentially and which comprisesan origin of replication, such as a plasmid, phage or cosmid, to whichanother nucleic acid segment may be attached so as to bring about thereplication of the attached segment. Cloning vectors may be capable ofreplication in one cell type and expression in another (“shuttlevector”).

Vectors may be introduced into the desired host cells by methods knownin the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; and Hartmutet al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

A polynucleotide according to the invention can also be introduced invivo by lipofection. For the past decade, there has been increasing useof liposomes for encapsulation and transfection of nucleic acids invitro. Synthetic cationic lipids designed to limit the difficulties anddangers encountered with liposome-mediated transfection can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Felgner et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84: 7413; Mackey,et al., 1988, Proc. Natl. Acad. Sci. U.S.A 85:8027-8031; and Ulmer etal., 1993, Science 259: 1745-1748). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Felgner andRingold, 1989, Science 337:387-388). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO95/18863 and WO96/17823, and in U.S.Pat. No. 5,459,127. The use of lipofection to introduce exogenous genesinto the specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. It is clear that directing transfection to particular celltypes would be particularly preferred in a tissue with cellularheterogeneity, such as pancreas, liver, kidney, and the brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting (Mackey, et al., 1988, supra). Targeted peptides, e.g.,hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO 95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., 1992, Hum. Gene Ther. 3: 147-154; and Wu and Wu, 1987, J. Biol.Chem. 262: 4429-4432).

The term “transfection” means the uptake of exogenous or heterologousRNA or DNA by a cell. A cell has been “transfected” by exogenous orheterologous RNA or DNA when such RNA or DNA has been introduced insidethe cell. A cell has been “transformed” by exogenous or heterologous RNAor DNA when the transfected RNA or DNA effects a phenotypic change. Thetransforming RNA or DNA can be integrated (covalently linked) intochromosomal DNA making up the genome of the cell.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The term “genetic region” will refer to a region of a nucleic acidmolecule or a nucleotide sequence that comprises a gene encoding apolypeptide.

In addition, the recombinant vector comprising a polynucleotideaccording to the invention may include one or more origins forreplication in the cellular hosts in which their amplification or theirexpression is sought, markers or selectable markers.

The term “selectable marker” means an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, colorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

The term “reporter gene” means a nucleic acid encoding an identifyingfactor that is able to be identified based upon the reporter gene'seffect, wherein the effect is used to track the inheritance of a nucleicacid of interest, to identify a cell or organism that has inherited thenucleic acid of interest, and/or to measure gene expression induction ortranscription. Examples of reporter genes known and used in the artinclude: luciferase (Luc), green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes may also beconsidered reporter genes.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. Promotersthat cause a gene to be expressed in a specific cell type are commonlyreferred to as “cell-specific promoters” or “tissue-specific promoters”.Promoters that cause a gene to be expressed at a specific stage ofdevelopment or cell differentiation are commonly referred to as“developmentally-specific promoters” or “cell differentiation-specificpromoters”. Promoters that are induced and cause a gene to be expressedfollowing exposure or treatment of the cell with an agent, biologicalmolecule, chemical, ligand, light, or the like that induces the promoterare commonly referred to as “inducible promoters” or “regulatablepromoters”. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease SI), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains introns) and translated intothe protein encoded by the coding sequence.

“Transcriptional and translational control sequences” are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

The term “response element” means one or more cis-acting DNA elementswhich confer responsiveness on a promoter mediated through interactionwith the DNA-binding domains of the first chimeric gene. This DNAelement may be either palindromic (perfect or imperfect) in its sequenceor composed of sequence motifs or half sites separated by a variablenumber of nucleotides. The half sites can be similar or identical andarranged as either direct or inverted repeats or as a single half siteor multimers of adjacent half sites in tandem. The response element maycomprise a minimal promoter isolated from different organisms dependingupon the nature of the cell or organism into which the response elementwill be incorporated. The DNA binding domain of the first hybrid proteinbinds, in the presence or absence of a ligand, to the DNA sequence of aresponse element to initiate or suppress transcription of downstreamgene(s) under the regulation of this response element. Examples of DNAsequences for response elements of the natural ecdysone receptorinclude: RRGG/=TCANTGAC/ACYY (see Cherbas L., et. al., (1991), GenesDev. 5, 120-131); AGGTCAN_((n))AGGTCA, where N_((n)) can be one or morespacer nucleotides (see D'Avino P P., et. al., (1995), Mol. Cell.Endocrinol, 113, 1-9); and GGGTTGAATGAATTT (see Antoniewski C., et. al.,(1994). Mol. Cell. Biol. 14, 4465-4474).

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid or polynucleotide. Expression may also refer to translationof m-RNA into a protein or polypeptide.

The terms “cassette”, “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

For purposes of this invention, the term “gene switch” refers to thecombination of a response element associated with a promoter, and anEcR-based system, which in the presence of one or more ligands,modulates the expression of a gene into which the response element andpromoter are incorporated.

The terms “modulate” and “modulates” mean to induce, reduce or inhibitnucleic acid or gene expression, resulting in the respective induction,reduction or inhibition of protein or polypeptide production.

The plasmids or vectors according to the invention may further compriseat least one promoter suitable for driving expression of a gene in ahost cell. The term “expression vector” means a vector, plasmid orvehicle designed to enable the expression of an inserted nucleic acidsequence following transformation into the host. The cloned gene, i.e.,the inserted nucleic acid sequence, is usually placed under the controlof control elements such as a promoter, a minimal promoter, an enhancer,or the like. Initiation control regions or promoters, which are usefulto drive expression of a nucleic acid in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to: viral promoters, bacterialpromoters, animal promoters, mammalian promoters, synthetic promoters,constitutive promoters, tissue specific promoter, developmental specificpromoters, inducible promoters, light regulated promoters; CYC1, HIS3,GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TPI, alkaline phosphatase promoters (useful for expression inSaccharomyces); AOX1 promoter (useful for expression in Pichia);β-lactamase, lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trcpromoters (useful for expression in Escherichia coli); light regulated-,seed specific-, pollen specific-, ovary specific-, pathogenesis ordisease related-, cauliflower mosaic virus 35S, CMV 35S minimal, cassayavein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase,stress inducible, rice tungro bacilliform virus, plant super-promoter,potato leucine aminopeptidase, nitrate reductase, mannopine synthase,nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters(useful for expression in plant cells); animal and mammalian promotersknown in the art include, but are not limited to, the SV40 early (SV40e)promoter region, the promoter contained in the 3′ long terminal repeat(LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or majorlate promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus(CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase(TK) promoter, a baculovirus IE1 promoter, an elongation factor 1 alpha(EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin(Ubc) promoter, an albumin promoter, the regulatory sequences of themouse metallothionein-L promoter and transcriptional control regions,the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and thelike), the promoters of the intermediate filaments (desmin,neurofilaments, keratin, GFAP, and the like), the promoters oftherapeutic genes (of the MDR, CFTR or factor VIII type, and the like),pathogenesis or disease related-promoters, and promoters that exhibittissue specificity and have been utilized in transgenic animals, such asthe elastase I gene control region which is active in pancreatic acinarcells; insulin gene control region active in pancreatic beta cells,immunoglobulin gene control region active in lymphoid cells, mousemammary tumor virus control region active in testicular, breast,lymphoid and mast cells; albumin gene, Apo AI and Apo AII controlregions active in liver, alpha-fetoprotein gene control region active inliver, alpha 1-antitrypsin gene control region active in the liver,beta-globin gene control region active in myeloid cells, myelin basicprotein gene control region active in oligodendrocyte cells in thebrain, myosin light chain-2 gene control region active in skeletalmuscle, and gonadotropic releasing hormone gene control region active inthe hypothalamus, pyruvate kinase promoter, villin promoter, promoter ofthe fatty acid binding intestinal protein, promoter of the smooth musclecell α-actin, and the like. In addition, these expression sequences maybe modified by addition of enhancer or regulatory sequences and thelike.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor 1 (EF1) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. Optionally, a termination site may be unnecessaryhowever, it is most preferred if included. In a preferred embodiment ofthe invention, the termination control region may be comprise or bederived from a synthetic sequence, synthetic polyadenylation signal, anSV40 late polyadenylation signal, an SV40 polyadenylation signal, abovine growth hormone (BGH) polyadenylation signal, viral terminatorsequences, or the like.

The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)”refer to DNA sequences located downstream (3′) of a coding sequence andmay comprise polyadenylation [poly(A)] recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

“Regulatory region” means a nucleic acid sequence that regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

A regulatory region from a “heterologous source” is a regulatory regionthat is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are designed by one having ordinary skill inthe art.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA,or other RNA that is not translated yet has an effect on cellularprocesses.

A “polypeptide” is a polymeric compound comprised of covalently linkedamino acid residues. Amino acids have the following general structure:

Amino acids are classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup. A polypeptide of the invention preferably comprises at leastabout 14 amino acids.

A “protein” is a polypeptide that performs a structural or functionalrole in a living cell.

An “isolated polypeptide” or “isolated protein” is a polypeptide orprotein that is substantially free of those compounds that are normallyassociated therewith in its natural state (e.g., other proteins orpolypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is notmeant to exclude artificial or synthetic mixtures with other compounds,or the presence of impurities which do not interfere with biologicalactivity, and which may be present, for example, due to incompletepurification, addition of stabilizers, or compounding into apharmaceutically acceptable preparation.

A “substitution mutant polypeptide” or a “substitution mutant” will beunderstood to mean a mutant polypeptide comprising a substitution of atleast one (1) wild-type or naturally occurring amino acid with adifferent amino acid relative to the wild-type or naturally occurringpolypeptide. A substitution mutant polypeptide may comprise only one (1)wild-type or naturally occurring amino acid substitution and may bereferred to as a “point mutant” or a “single point mutant” polypeptide.Alternatively, a substitution mutant polypeptide may comprise asubstitution of two (2) or more wild-type or naturally occurring aminoacids with 2 or more amino acids relative to the wild-type ornaturally-occurring polypeptide. According to the invention, a Group Hnuclear receptor ligand binding domain polypeptide comprising asubstitution mutation comprises a substitution of at least one (1)wild-type or naturally occurring amino acid with a different amino acidrelative to the wild-type or naturally occurring Group H nuclearreceptor ligand binding domain polypeptide.

Wherein the substitution mutant polypeptide comprises a substitution oftwo (2) or more wild-type or naturally occurring amino acids, thissubstitution may comprise either an equivalent number of wild-type ornaturally occurring amino acids deleted for the substitution, i.e., 2wild-type or naturally occurring amino acids replaced with 2non-wild-type or non-naturally occurring amino acids, or anon-equivalent number of wild-type amino acids deleted for thesubstitution, i.e., 2 wild-type amino acids replaced with 1non-wild-type amino acid (a substitution+deletion mutation), or 2wild-type amino acids replaced with 3 non-wild-type amino acids (asubstitution+insertion mutation). Substitution mutants may be describedusing an abbreviated nomenclature system to indicate the amino acidresidue and number replaced within the reference polypeptide sequenceand the new substituted amino acid residue. For example, a substitutionmutant in which the twentieth (20^(th)) amino acid residue of apolypeptide is substituted may be abbreviated as “x20z”, wherein “x” isthe amino acid to be replaced, “20” is the amino acid residue positionor number within the polypeptide, and “z” is the new substituted aminoacid.

Therefore, a substitution mutant abbreviated interchangeably as “E20A”or “Glu20Ala” indicates that the mutant comprises an alanine residue(commonly abbreviated in the art as “A” or “Ala”) in place of theglutamic acid (commonly abbreviated in the art as “E” or “Glu”) atposition 20 of the polypeptide. A mutation or mutant can be any change,including but not limited to substitutions, deletions, insertions, orany combination thereof.

A substitution mutation may be made by any technique for mutagenesisknown in the art, including but not limited to, in vitro site-directedmutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253: 6551;Zoller and Smith, 1984, DNA 3: 479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 710),use of TAB® linkers (Pharmacia), restriction endonucleasedigestion/fragment deletion and substitution,PCR-mediated/oligonucleotide-directed mutagenesis, and the like.PCR-based techniques are preferred for site-directed mutagenesis (seeHiguchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology:Principles and Applications for DNA Amplification, H. Erlich, ed.,Stockton Press, Chapter 6, pp. 61-70).

“Fragment” of a polypeptide according to the invention will beunderstood to mean a polypeptide whose amino acid sequence is shorterthan that of the reference polypeptide and which comprises, over theentire portion with these reference polypeptides, an identical aminoacid sequence. Such fragments may, where appropriate, be included in alarger polypeptide of which they are a part. Such fragments of apolypeptide according to the invention may have a length of at least 2,3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30,35, 40, 45, 50, 100, 200, 240, or 300 amino acids.

A “variant” of a polypeptide or protein is any analogue, fragment,derivative, or mutant which is derived from a polypeptide or protein andwhich retains at least one biological property of the polypeptide orprotein. Different variants of the polypeptide or protein may exist innature. These variants may be allelic variations characterized bydifferences in the nucleotide sequences of the structural gene codingfor the protein, or may involve differential splicing orpost-translational modification. The skilled artisan can producevariants having single or multiple amino acid substitutions, deletions,additions, or replacements. These variants may include, inter alia: (a)variants in which one or more amino acid residues are substituted withconservative or non-conservative amino acids, (b) variants in which oneor more amino acids are added to the polypeptide or protein, (c)variants in which one or more of the amino acids includes a substituentgroup, and (d) variants in which the polypeptide or protein is fusedwith another polypeptide such as serum albumin. The techniques forobtaining these variants, including genetic (suppressions, deletions,mutations, etc.), chemical, and enzymatic techniques, are known topersons having ordinary skill in the art. A variant polypeptidepreferably comprises at least about 14 amino acids.

A “heterologous protein” refers to a protein not naturally produced inthe cell.

A “mature protein” refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

The term “signal peptide” refers to an amino terminal polypeptidepreceding the secreted mature protein. The signal peptide is cleavedfrom and is therefore not present in the mature protein. Signal peptideshave the function of directing and translocating secreted proteinsacross cell membranes. Signal peptide is also referred to as signalprotein.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

The term “homology” refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown to the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions that form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s) and size determination of thedigested fragments.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., 1987, Cell 50: 667). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity. However, in common usage and in the instant application, theterm “homologous,” when modified with an adverb such as “highly,” mayrefer to sequence similarity and not a common evolutionary origin.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., 1987, Cell 50:667).

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., 1989, supra.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotide bases that donot substantially affect the functional properties of the resultingtranscript. It is therefore understood that the invention encompassesmore than the specific exemplary sequences. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, the skilled artisan recognizes that substantially similarsequences encompassed by this invention are also defined by theirability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65°C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), withthe sequences exemplified herein. Substantially similar nucleic acidfragments of the instant invention are those nucleic acid fragmentswhose DNA sequences are at least 70% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Preferred substantiallynucleic acid fragments of the instant invention are those nucleic acidfragments whose DNA sequences are at least 80% identical to the DNAsequence of the nucleic acid fragments reported herein. More preferrednucleic acid fragments are at least 90% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Even more preferred arenucleic acid fragments that are at least 95% identical to the DNAsequence of the nucleic acid fragments reported herein.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than about 40% of the amino acidsare identical, or greater than 60% are similar (functionally identical).Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215: 403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesmay be performed using the Clustal method of alignment (Higgins andSharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method may be selected: KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215: 403-410(1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715USA). Within the context of this application it will be understood thatwhere sequence analysis software is used for analysis, that the resultsof the analysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters, which originally load withthe software when first initialized.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

As used herein, two or more individually operable gene regulationsystems are said to be “orthogonal” when; a) modulation of each of thegiven systems by its respective ligand, at a chosen concentration,results in a measurable change in the magnitude of expression of thegene of that system, and b) the change is statistically significantlydifferent than the change in expression of all other systemssimultaneously operable in the cell, tissue, or organism, regardless ofthe simultaneity or sequentially of the actual modulation. Preferably,modulation of each individually operable gene regulation system effectsa change in gene expression at least 2-fold greater than all otheroperable systems in the cell, tissue, or organism. More preferably, thechange is at least 5-fold greater. Even more preferably, the change isat least 10-fold greater. Still more preferably, the change is at least100 fold greater. Even still more preferably, the change is at least500-fold greater. Ideally, modulation of each of the given systems byits respective ligand at a chosen concentration results in a measurablechange in the magnitude of expression of the gene of that system and nomeasurable change in expression of all other systems operable in thecell, tissue, or organism. In such cases the multiple inducible generegulation system is said to be “fully orthogonal”. The presentinvention is useful to search for orthogonal ligands and orthogonalreceptor-based gene expression systems such as those described inco-pending U.S. application Ser. No. 09/965,697, which is incorporatedherein by reference in its entirety.

Gene Expression Modulation System of the Invention

Applicants have identified herein amino acid residues that are involvedin ligand binding to a Group H nuclear receptor ligand binding domainthat affect the ligand sensitivity and magnitude of induction in anecdysone receptor-based inducible gene expression system. Applicantsdescribe herein the construction of Group H nuclear receptors thatcomprise substitution mutations (referred to herein as “substitutionmutants”) at these critical residues and the demonstration that thesesubstitution mutant nuclear receptors are useful in methods ofmodulating gene expression. As presented herein, Applicants' novelsubstitution mutant nuclear receptors and their use in a nuclearreceptor-based inducible gene expression system provides an improvedinducible gene expression system in both prokaryotic and eukaryotic hostcells in which ligand sensitivity and magnitude of transactivation maybe selected as desired, depending upon the application.

Thus, the present invention relates to novel substitution mutant Group Hnuclear receptor polynucleotides and polypeptides, a nuclearreceptor-based inducible gene expression system comprising such mutatedGroup H nuclear receptor polynucleotides and polypeptides, and methodsof modulating the expression of a gene within a host cell using such anuclear receptor-based inducible gene expression system.

In particular, the present invention relates to a gene expressionmodulation system comprising at least one gene expression cassette thatis capable of being expressed in a host cell comprising a polynucleotidethat encodes a polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation. Preferably, the GroupH nuclear receptor ligand binding domain comprising a substitutionmutation is from an ecdysone receptor, a ubiquitous receptor, an orphanreceptor 1, a NER-1, a steroid hormone nuclear receptor 1, a retinoid Xreceptor interacting protein −15, a liver X receptor β, a steroidhormone receptor like protein, a liver X receptor, a liver X receptor α,a farnesoid X receptor, a receptor interacting protein 14, and afarnesol receptor. More preferably, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is from an ecdysonereceptor.

In a specific embodiment, the gene expression modulation systemcomprises a gene expression cassette comprising a polynucleotide thatencodes a polypeptide comprising a transactivation domain, a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another specific embodiment, the gene expression modulation systemcomprises a gene expression cassette comprising a) a polynucleotide thatencodes a polypeptide comprising a transactivation domain, a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, and b) a secondnuclear receptor ligand binding domain selected from the groupconsisting of a vertebrate retinoid X receptor ligand binding domain, aninvertebrate retinoid X receptor ligand binding domain, an ultraspiracleprotein ligand binding domain, and a chimeric ligand binding domaincomprising two polypeptide fragments, wherein the first polypeptidefragment is from a vertebrate retinoid X receptor ligand binding domain,an invertebrate retinoid X receptor ligand binding domain, or anultraspiracle protein ligand binding domain, and the second polypeptidefragment is from a different vertebrate retinoid X receptor ligandbinding domain, invertebrate retinoid X receptor ligand binding domain,or ultraspiracle protein ligand binding domain. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another specific embodiment, the gene expression modulation systemcomprises a first gene expression cassette comprising a polynucleotidethat encodes a first polypeptide comprising a DNA-binding domain thatrecognizes a response element associated with a gene whose expression isto be modulated and a nuclear receptor ligand binding domain, and asecond gene expression cassette comprising a polynucleotide that encodesa second polypeptide comprising a transactivation domain and a nuclearreceptor ligand binding domain, wherein one of the nuclear receptorligand binding domains is a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation. In a preferred embodiment,the first polypeptide is substantially free of a transactivation domainand the second polypeptide is substantially free of a DNA bindingdomain. For purposes of the invention, “substantially free” means thatthe protein in question does not contain a sufficient sequence of thedomain in question to provide activation or binding activity. The geneexpression modulation system may further comprise a third geneexpression cassette comprising: i) a response element recognized by theDNA-binding domain of the first polypeptide of the first gene expressioncassette; ii) a promoter that is activated by the transactivation domainof the second polypeptide of the second gene expression cassette; andiii) a gene whose expression is to be modulated.

Wherein when only one nuclear receptor ligand binding domain is a GroupH ligand binding domain comprising a substitution mutation, the othernuclear receptor ligand binding domain may be from any other nuclearreceptor that forms a dimer with the Group H ligand binding domaincomprising the substitution mutation. For example, when the Group Hnuclear receptor ligand binding domain comprising a substitutionmutation is an ecdysone receptor ligand binding domain comprising asubstitution mutation, the other nuclear receptor ligand binding domain(“partner”) may be from an ecdysone receptor, a vertebrate retinoid Xreceptor (RXR), an invertebrate RXR, an ultraspiracle protein (USP), ora chimeric nuclear receptor comprising at least two different nuclearreceptor ligand binding domain polypeptide fragments selected from thegroup consisting of a vertebrate RXR, an invertebrate RXR, and a RXR(see co-pending applications PCT/US01/09050, U.S. 60/294,814, and U.S.60/294,819, incorporated herein by reference in their entirety). The“partner” nuclear receptor ligand binding domain may further comprise atruncation mutation, a deletion mutation, a substitution mutation, oranother modification.

Preferably, the vertebrate RXR ligand binding domain is from a humanHomo sapiens, mouse Mus musculus, rat Rattus norvegicus, chicken Gallusgallus, pig Sus scrofa domestica, frog Xenopus laevis, zebrafish Daniorerio, tunicate Polyandrocarpa misakiensis, orjellyfish Tripedaliacysophora RXR.

Preferably, the invertebrate RXR ligand binding domain is from a locustLocusta migratoria RXR polypeptide (“LmRXR”), an ixodid tick Amblyommaamericanum RXR homolog 1 (“AmaRX1”), a ixodid tick Amblyomma americanumRXR homolog 2 (“AmaRXR2”), a fiddler crab Celuca pugilator RXR homolog(“CpRXR”), a beetle Tenebrio molitor RXR homolog (“TmRXR”), a honeybeeApis mellifera RXR homolog (“AmRXR”), an aphid Myzus persicae RXRhomolog (“MpRXR”), or a non-Dipteran/non-Lepidopteran RXR homolog.

Preferably, the chimeric RXR ligand binding domain comprises at leasttwo polypeptide fragments selected from the group consisting of avertebrate species RXR polypeptide fragment, an invertebrate species RXRpolypeptide fragment, and a non-Dipteran/non-Lepidopteran invertebratespecies RXR homolog polypeptide fragment. A chimeric RXR ligand bindingdomain for use in the present invention may comprise at least twodifferent species RXR polypeptide fragments, or when the species is thesame, the two or more polypeptide fragments may be from two or moredifferent isoforms of the species RXR polypeptide fragment.

In a preferred embodiment, the chimeric RXR ligand binding domaincomprises at least one vertebrate species RXR polypeptide fragment andone invertebrate species RXR polypeptide fragment.

In a more preferred embodiment, the chimeric RXR ligand binding domaincomprises at least one vertebrate species RXR polypeptide fragment andone non-Dipteran/non-Lepidopteran invertebrate species RXR homologpolypeptide fragment.

In a specific embodiment, the gene whose expression is to be modulatedis a homologous gene with respect to the host cell. In another specificembodiment, the gene whose expression is to be modulated is aheterologous gene with respect to the host cell.

The ligands for use in the present invention as described below, whencombined with the ligand binding domain of the nuclear receptor(s),which in turn are bound to the response element linked to a gene,provide the means for external temporal regulation of expression of thegene. The binding mechanism or the order in which the various componentsof this invention bind to each other, that is, for example, ligand toligand binding domain, DNA-binding domain to response element,transactivation domain to promoter, etc., is not critical.

In a specific example, binding of the ligand to the ligand bindingdomain of a Group H nuclear receptor and its nuclear receptor ligandbinding domain partner enables expression or suppression of the gene.This mechanism does not exclude the potential for ligand binding to theGroup H nuclear receptor (GHNR) or its partner, and the resultingformation of active homodimer complexes (e.g. GHNR+GHNR orpartner+partner). Preferably, one or more of the receptor domains isvaried producing a hybrid gene switch. Typically, one or more of thethree domains, DBD, LBD, and transactivation domain, may be chosen froma source different than the source of the other domains so that thehybrid genes and the resulting hybrid proteins are optimized in thechosen host cell or organism for transactivating activity, complementarybinding of the ligand, and recognition of a specific response element.In addition, the response element itself can be modified or substitutedwith response elements for other DNA binding protein domains such as theGAL4 protein from yeast (see Sadowski, et al. (1988), Nature, 335:563-564) or LexA protein from Escherichia coli (see Brent and Ptashne(1985), Cell, 43: 729-736), or synthetic response elements specific fortargeted interactions with proteins designed, modified, and selected forsuch specific interactions (see, for example, Kim, et al. (1997), Proc.Natl. Acad. Sci., USA, 94:3 616-3620) to accommodate hybrid receptors.Another advantage of two-hybrid systems is that they allow choice of apromoter used to drive the gene expression according to a desired endresult. Such double control can be particularly important in areas ofgene therapy, especially when cytotoxic proteins are produced, becauseboth the timing of expression as well as the cells wherein expressionoccurs can be controlled. When genes, operably linked to a suitablepromoter, are introduced into the cells of the subject, expression ofthe exogenous genes is controlled by the presence of the system of thisinvention. Promoters may be constitutively or inducibly regulated or maybe tissue-specific (that is, expressed only in a particular type ofcells) or specific to certain developmental stages of the organism.

The ecdysone receptor is a member of the nuclear receptor superfamilyand classified into subfamily 1, group H (referred to herein as “Group Hnuclear receptors”). The members of each group share 40-60% amino acididentity in the E (ligand binding) domain (Laudet et al., A UnifiedNomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97:161-163). In addition to the ecdysone receptor, other members of thisnuclear receptor subfamily 1, group H include: ubiquitous receptor (UR),orphan receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1),retinoid X receptor interacting protein −15 (RIP-15), liver X receptor β(LXRβ), steroid hormone receptor like protein (RLD-1), liver X receptor(LXR), liver X receptor α (LXRα, farnesoid X receptor (FXR), receptorinteracting protein 14 (RIP-14), and farnesol receptor (HRR-1).

Applicants have developed a CfEcR homology model and have used thishomology model together with a published Chironomous tetans ecdysonereceptor (“CtEcR”) homology model (Wurtz et al., 2000) to identifycritical residues involved in binding to ecdysteroids andnon-ecdysteroids. The synthetic non-ecdysteroids, diacylhydrazines, havebeen shown to bind lepidopteran EcRs with high affinity and induceprecocious incomplete molt in these insects (Wing et al., 1988) andseveral of these compounds are currently marketed as insecticides. Theligand binding cavity or “pocket” of EcRs has evolved to fit the longbackbone structures of ecdysteroids such as 20-hydroxyecdysone (20E).The diacylhydrazines have a compact structure compared to ecdysteroidsand occupy only the bottom part of the EcR binding pocket. This leaves afew critical residues at the top part of the binding pocket that makecontact with ecdysteroids but not with non-ecdysteroids such asbisacylhydrazines. Applicants describe herein the construction of mutantecdysone receptors comprising a substitution mutation at these bindingpocket residues and have identified several classes of substitutionmutant ecdysone receptors with modified ligand binding andtransactivation characteristics.

Given the close relatedness of ecdysone receptor to other Group Hnuclear receptors, Applicants' identified ecdysone receptor ligandbinding domain substitution mutations are also expected to work whenintroduced into the analogous position of the ligand binding domains ofother Group H nuclear receptors to modify their ligand binding or ligandsensitivity. One of skill in the art can identify analogous amino acidpositions by sequence and function using routine methods in the art suchas sequence analysis, analysis of the binding pocket through homologymodeling and binding assays. Applicants' novel substitution mutatedGroup H nuclear receptor polynucleotides and polypeptides are useful ina nuclear receptor-based inducible gene modulation system for variousapplications including gene therapy, expression of proteins of interestin host cells, production of transgenic organisms, and cell-basedassays.

In particular, Applicants describe herein a novel gene expressionmodulation system comprising a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation. This gene expression systemmay be a “single switch”-based gene expression system in which thetransactivation domain, DNA-binding domain and ligand binding domain areon one encoded polypeptide. Alternatively, the gene expressionmodulation system may be a “dual switch”- or “two-hybrid”-based geneexpression modulation system in which the transactivation domain andDNA-binding domain are located on two different encoded polypeptides.Applicants have demonstrated for the first time that a substitutionmutated nuclear receptor can be used as a component of a nuclearreceptor-based inducible gene expression system to modify ligand bindingactivity and/or ligand specificity in both prokaryotic and eukaryoticcells. As discussed herein, Applicants' findings are both unexpected andsurprising.

An ecdysone receptor-based gene expression modulation system of thepresent invention may be either heterodimeric or homodimeric. Afunctional EcR complex generally refers to a heterodimeric proteincomplex consisting of two members of the steroid receptor family, anecdysone receptor protein obtained from various insects, and anultraspiracle (USP) protein or the vertebrate homolog of USP, retinoid Xreceptor protein (see Yao, et al. (1993) Nature 366: 476-479; Yao, etal., (1992) Cell 71: 63-72). However, the complex may also be ahomodimer as detailed below. The functional ecdysteroid receptor complexmay also include additional protein(s) such as immunophilins. Additionalmembers of the steroid receptor family of proteins, known astranscriptional factors (such as DHR38 or betaFTZ-1), may also be liganddependent or independent partners for EcR, USP, and/or RXR.Additionally, other cofactors may be required such as proteins generallyknown as coactivators (also termed adapters or mediators). Theseproteins do not bind sequence-specifically to DNA and are not involvedin basal transcription. They may exert their effect on transcriptionactivation through various mechanisms, including stimulation ofDNA-binding of activators, by affecting chromatin structure, or bymediating activator-initiation complex interactions. Examples of suchcoactivators include RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1,TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the promiscuouscoactivator C response element B binding protein, CBP/p300 (for reviewsee Glass et al., Curr. Opin. Cell Biol. 9: 222-232, 1997). Also,protein cofactors generally known as corepressors (also known asrepressors, silencers, or silencing mediators) may be required toeffectively inhibit transcriptional activation in the absence of ligand.These corepressors may interact with the unliganded ecdysone receptor tosilence the activity at the response element. Current evidence suggeststhat the binding of ligand changes the conformation of the receptor,which results in release of the corepressor and recruitment of the abovedescribed coactivators, thereby abolishing their silencing activity.Examples of corepressors include N-CoR and SMRT (for review, see Horwitzet al. Mol. Endocrinol. 10: 1167-1177, 1996). These cofactors may eitherbe endogenous within the cell or organism, or may be added exogenouslyas transgenes to be expressed in either a regulated or unregulatedfashion. Homodimer complexes of the ecdysone receptor protein, USP, orRXR may also be functional under some circumstances.

The ecdysone receptor complex typically includes proteins that aremembers of the nuclear receptor superfamily wherein all members aregenerally characterized by the presence of an amino-terminaltransactivation domain, a DNA binding domain (“DBD”), and a ligandbinding domain (“LBD”) separated from the DBD by a hinge region. As usedherein, the term “DNA binding domain” comprises a minimal polypeptidesequence of a DNA binding protein, up to the entire length of a DNAbinding protein, so long as the DNA binding domain functions toassociate with a particular response element. Members of the nuclearreceptor superfamily are also characterized by the presence of four orfive domains: A/B, C, D, E, and in some members F (see U.S. Pat. No.4,981,784 and Evans, Science 240:889-895 (1988)). The “A/B” domaincorresponds to the transactivation domain, “C” corresponds to the DNAbinding domain, “D” corresponds to the hinge region, and “E” correspondsto the ligand binding domain. Some members of the family may also haveanother transactivation domain on the carboxy-terminal side of the LBDcorresponding to “F”.

The DBD is characterized by the presence of two cysteine zinc fingersbetween which are two amino acid motifs, the P-box and the D-box, whichconfer specificity for ecdysone response elements. These domains may beeither native, modified, or chimeras of different domains ofheterologous receptor proteins. The EcR receptor, like a subset of thesteroid receptor family, also possesses less well-defined regionsresponsible for heterodimerization properties. Because the domains ofnuclear receptors are modular in nature, the LBD, DBD, andtransactivation domains may be interchanged.

Gene switch systems are known that incorporate components from theecdysone receptor complex. However, in these known systems, whenever EcRis used it is associated with native or modified DNA binding domains andtransactivation domains on the same molecule. USP or RXR are typicallyused as silent partners. Applicants have previously shown that when DNAbinding domains and transactivation domains are on the same molecule thebackground activity in the absence of ligand is high and that suchactivity is dramatically reduced when DNA binding domains andtransactivation domains are on different molecules, that is, on each oftwo partners of a heterodimeric or homodimeric complex (seePCT/US01/09050).

Gene Expression Cassettes of the Invention

The novel nuclear receptor-based inducible gene expression system of theinvention comprises at least one gene expression cassette that iscapable of being expressed in a host cell, wherein the gene expressioncassette comprises a polynucleotide that encodes a polypeptidecomprising a Group H nuclear receptor ligand binding domain comprising asubstitution mutation. Thus, Applicants' invention also provides novelgene expression cassettes for use in the gene expression system of theinvention.

In a specific embodiment, the gene expression cassette that is capableof being expressed in a host cell comprises a polynucleotide thatencodes a polypeptide selected from the group consisting of a) apolypeptide comprising a transactivation domain, a DNA-binding domain,and a Group H nuclear receptor ligand binding domain comprising asubstitution mutation; b) a polypeptide comprising a DNA-binding domainand a Group H nuclear receptor ligand binding domain comprising asubstitution mutation; and c) a polypeptide comprising a transactivationdomain and a Group H nuclear receptor ligand binding domain comprising asubstitution mutation.

In another specific embodiment, the present invention provides a geneexpression cassette that is capable of being expressed in a host cell,wherein the gene expression cassette comprises a polynucleotide thatencodes a hybrid polypeptide selected from the group consisting of a) ahybrid polypeptide comprising a transactivation domain, a DNA-bindingdomain, and a Group H nuclear receptor ligand binding domain comprisinga substitution mutation; b) a hybrid polypeptide comprising aDNA-binding domain and a Group H nuclear receptor ligand binding domaincomprising a substitution mutation; and c) a hybrid polypeptidecomprising a transactivation domain and a Group H nuclear receptorligand binding domain comprising a substitution mutation. A hybridpolypeptide according to the invention comprises at least twopolypeptide fragments, wherein each polypeptide fragment is from adifferent source, i.e., a different polypeptide, a different nuclearreceptor, a different species, etc. The hybrid polypeptide according tothe invention may comprise at least two polypeptide domains, whereineach polypeptide domain is from a different source.

In a specific embodiment, the Group H nuclear receptor ligand bindingdomain comprising a substitution mutation is from an ecdysone receptor,a ubiquitous receptor, an orphan receptor 1, a NER-1, a steroid hormonenuclear receptor 1, a retinoid X receptor interacting protein −15, aliver X receptor β, a steroid hormone receptor like protein, a liver Xreceptor, a liver X receptor a, a farnesoid X receptor, a receptorinteracting protein 14, and a farnesol receptor. In a preferredembodiment, the Group H nuclear receptor ligand binding domain is froman ecdysone receptor.

Thus, the present invention also provides a gene expression cassettecomprising a polynucleotide that encodes a polypeptide selected from thegroup consisting of a) a polypeptide comprising a transactivationdomain, a DNA-binding domain, and an ecdysone receptor ligand bindingdomain comprising a substitution mutation; b) a polypeptide comprising aDNA-binding domain and an ecdysone receptor ligand binding domaincomprising a substitution mutation; and c) a polypeptide comprising atransactivation domain and an ecdysone receptor ligand binding domaincomprising a substitution mutation. Preferably, the gene expressioncassette comprises a polynucleotide that encodes a hybrid polypeptideselected from the group consisting of a) a hybrid polypeptide comprisinga transactivation domain, a DNA-binding domain, and an ecdysone receptorligand binding domain comprising a substitution mutation; b) a hybridpolypeptide comprising a DNA-binding domain and an ecdysone receptorligand binding domain comprising a substitution mutation; and c) ahybrid polypeptide comprising a transactivation domain and an ecdysonereceptor ligand binding domain comprising a substitution mutation;wherein the encoded hybrid polypeptide comprises at least twopolypeptide fragments, wherein each polypeptide fragment is from adifferent source.

The ecdysone receptor (EcR) ligand binding domain (LBD) may be from aninvertebrate EcR, preferably selected from the class Arthropod EcR.Preferably the EcR is selected from the group consisting of aLepidopteran EcR, a Dipteran EcR, an Orthopteran EcR, a Homopteran EcRand a Hemipteran EcR. More preferably, the EcR ligand binding domain foruse in the present invention is from a spruce budworm Choristoneurafumiferana EcR (“CfEcR”), a beetle Tenebrio molitor EcR (“TmEcR”), aManduca sexta EcR (“MsEcR”), a Heliothies virescens EcR (“HvEcR”), amidge Chironomus tentans EcR (“CtEcR”), a silk moth Bombyx mori EcR(“BmEcR”), a squinting bush brown Bicyclus anynana EcR (“BanEcR”), abuckeye Junonia coenia EcR (“JcEcR”), a fruit fly Drosophilamelanogaster EcR (“DmEcR”), a mosquito Aedes aegypti EcR (“AaEcR”), ablowfly Lucilia capitata (“LcEcR”), a blowfly Lucilia cuprina EcR(“LucEcR”), a blowfly Calliphora vicinia EcR (“CvEcR”), a Mediterraneanfruit fly Ceratitis capitata EcR (“CcEcR”), a locust Locusta migratoriaEcR (“LmEcR”), an aphid Myzus persicae EcR (“MpEcR”), a fiddler crabCeluca pugilator EcR (“CpEcR”), an ixodid tick Amblyomma americanum EcR(“AmaEcR”), a whitefly Bamecia argentifoli EcR (“BaEcR”) or a leafhopperNephotetix cincticeps EcR (“NcEcR”). More preferably, the LBD is from aCfEcR, a DmEcR, or an AmaEcR.

In a specific embodiment, the LBD is from a truncated EcR polypeptide.The EcR polypeptide truncation results in a deletion of at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, or 265 amino acids. Preferably, the EcRpolypeptide truncation results in a deletion of at least a partialpolypeptide domain. More preferably, the EcR polypeptide truncationresults in a deletion of at least an entire polypeptide domain. In aspecific embodiment, the EcR polypeptide truncation results in adeletion of at least an A/B-domain, a C-domain, a D-domain, an F-domain,an A/B/C-domains, an A/B/1/2-C-domains, an A/B/C/D-domains, anA/B/C/D/F-domains, an A/B/F-domains, an A/B/C/F-domains, a partial Edomain, or a partial F domain. A combination of several complete and/orpartial domain deletions may also be performed.

In a specific embodiment, the Group H nuclear receptor ligand bindingdomain is encoded by a polynucleotide comprising a codon mutation thatresults in a substitution of a) amino acid residue 48, 51, 52, 54, 92,95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, or 238 of SEQID NO: 1, b) amino acid residues 96 and 119 of SEQ ID NO: 1, c) aminoacid residues 110 and 128 of SEQ ID NO: 1, d) amino acid residues 52 and110 of SEQ ID NO: 1, e) amino acid residues 107, 110, and 127 of SEQ IDNO: 1, or f) amino acid residues 52, 107 and 127 of SEQ ID NO: 1. Inanother embodiment, the Group H nuclear receptor ligand binding domainis encoded by a polynucleotide comprising codon mutations that resultsin substitution of amino acid residues 107 and 127 and insertion ofamino acid 259 of SEQ ID NO: 1. In a preferred embodiment, the Group Hnuclear receptor ligand binding domain is from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain is encoded by a polynucleotide comprising a codonmutation that results in a substitution of a) an asparagine, arginine,tyrosine, tryptophan, leucine or lysine residue at a position equivalentto analogous to amino acid residue 48 of SEQ ID NO: 1, b) a methionine,asparagines or leucine residue at a position equivalent or analogous toamino acid residue 51 of SEQ ID NO: 1, c) a leucine, proline,methionine, arginine, tryptophan, glycine, glutamine or glutamic acidresidue at a position equivalent or analogous to amino acid residue 52of SEQ ID NO: 1, d) a tryptophan or threonine at a position equivalentor analogous to amino acid 54 of SEQ ID NO: 1, e) a leucine or glutamicacid at a position equivalent or analogous to amino acid 92 of SEQ IDNO: 1, f) a histidine, methionine or tryptophan residue at a positionequivalent or analogous to amino acid residue 95 of SEQ ID NO: 1, g) aleucine, serine, glutamic acid or tryptophan residue at a positionequivalent or analogous to amino acid residue 96 of SEQ ID NO: 1, h) atryptophan, proline, leucine, methionine or asparagine at a positionequivalent or analogous to amino acid 109 of SEQ ID NO: 1, i) a glutamicacid, tryptophan or asparagine residue at a position equivalent oranalogous to amino acid residue 110 of SEQ ID NO: 1, j) a phenylalanineat a position equivalent or analogous to amino acid 119 of SEQ ID NO: 1,k) a tryptophan or methionine at a position equivalent or analogous toamino acid 120 of SEQ ID NO: 1, 1) a glutamic acid, proline, leucine,cysteine, tryptophan, glycine, isoleucine, asparagine, serine, valine orarginine at a position equivalent or analogous to amino acid 125 of SEQID NO: 1, m) a phenylalanine at a position equivalent or analogous toamino acid 128 of SEQ ID NO: 1, n) a methionine, asparagine, glutamicacid or valine at a position equivalent or analogous to amino acid 132of SEQ ID NO: 1, o) an alanine, lysine, tryptophan or tyrosine residueat a position equivalent or analogous to amino acid residue 219 of SEQID NO: 1, p) a lysine, arginine or tyrosine residue at a positionequivalent or analogous to amino acid residue 223 of SEQ ID NO: 1, q) amethionine, arginine, tryptophan or isoleucine at a position equivalentor analogous to amino acid 234 of SEQ ID NO: 1, r) a proline, glutamicacid, leucine, methionine or tyrosine at a position equivalent oranalogous to amino acid 238 of SEQ ID NO: 1, s) a phenylalanine residueat a position equivalent or analogous to amino acid 119 of SEQ ID NO: 1and a threonine at a position equivalent or analogous to amino acid 96of SEQ ID NO: 1, t) a proline residue at a position equivalent oranalogous to amino acid 110 of SEQ ID NO: 1 and a phenylalanine residueat a position equivalent or analogous to amino acid 128 of SEQ ID NO: 1,u) a valine residue at a position equivalent or analogous to amino acid52 of SEQ ID NO: 1 and a praline residue at a position equivalent oranalogous to amino acid 110 of SEQ ID NO: 1, v) an isoleucine residue ata position equivalent or analogous to amino acid 107 of SEQ ID NO: 1, aglutamic acid residue at a position equivalent or analogous to aminoacid 127 of SEQ ID NO: 1 and a proline residue at a position equivalentor analogous to amino acid 110 of SEQ ID NO: 1, or w) an isoleucine at aposition equivalent or analogous to amino acid 107 of SEQ ID NO: 1, aglutamic acid at a position equivalent or analogous to amino acid 127 ofSEQ ID NO: 1 and a valine at a position equivalent or analogous to aminoacid 52 of SEQ ID NO: 1. In another embodiment, the Group H nuclearreceptor ligand binding domain is encoded by a polynucleotide comprisingcodon mutations that results in substitution of an isoleucine residue ata position equivalent or analogous to amino acid 107 of SEQ ID NO: 1, aglutamic acid residue at a position equivalent or analogous to aminoacid 127 of SEQ ID NO: 1 and insertion of a glycine residue at aposition equivalent or analogous to amino acid 259 of SEQ ID NO: 1. In apreferred embodiment, the Group H nuclear receptor ligand binding domainis from an ecdysone receptor.

In a specific embodiment, the Group H nuclear receptor ligand bindingdomain comprising a substitution mutation is an ecdysone receptor ligandbinding domain comprising a substitution mutation encoded by apolynucleotide comprising a codon mutation that results in asubstitution mutation selected from the group consisting of F48Y, F48W,F48L, F48N, F48R, F48K, I51M, I51N, I51L, T52M, T52V, T52L, T52E, T52P,T52R, T52W, T52G, T52Q, M54W, M54T, M92L, M92E, R95H, R95M, R95W, V96L,V96W, V96S, V96E, F109W, F109P, F109L, F109M, F109N, A110E, A110N,A110W, N119F, Y120W, Y120M, M125P, M125R, M125E, M125L, M125C, M125W,M125G, M125I, M125N, M125S, M125V, V128F, L132M, L132N, L132V, L132E,M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L2341, L234R,L234W, W238P, W238E, W238Y, W238M, W238L, N119F/V96T, V128F/A110P,T52V/A110P, V1071/Y127E/T52V, and V107/Y127E/A110P substitution mutationof SEQ ID NO: 1. In another specific embodiment, the Group H nuclearreceptor ligand binding domain comprising a substitution mutation is anecdysone receptor ligand binding domain comprising a substitutionmutation encoded by a polynucleotide comprising a codon mutation thatresults in substitution mutation V107I/Y127E of SEQ ID NO: 1, whichfurther comprises insertion mutation G259 of SEQ ID NO: 1(V107I/Y127E/G259).

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain polypeptide comprising a substitutionmutation encoded by a polynucleotide that hybridizes to a polynucleotidecomprising a codon mutation that results in a substitution mutationselected from the group consisting of F48Y, F48W, F48L, F48N, F48R,F48K, I51M, I51N, I51L, T52M, T52V, T52L, T52E, T52P, T52R, T52W, T52G,T52Q, M54W, M54T, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E,F109W, F109P, F109L, F109M, F109N, A110E, A110N, A110W, N119F, Y120W,Y120M, M125P, M125R, M125E, M125L, M125C, M125W, M125G, M1251, M125N,M125S, M125V, V128F, L132M, L132N, L132V, L132E, M219K, M219W, M219Y,M219A, L223K, L223R, L223Y, L234M, L2341, L234R, L234W, W238P, W238E,W238Y, W238M, W238L, N 19F/V96T, V128F/A110P, T52V/A110P,V107I/Y127E/T52V, and V107I/Y127E/A110P of SEQ ID NO: 1 underhybridization conditions comprising a hybridization step in less than500 mM salt and at least 37 degrees Celsius, and a washing step in2×SSPE at least 63 degrees Celsius. In a preferred embodiment, thehybridization conditions comprise less than 200 mM salt and at least 37degrees Celsius for the hybridization step. In another preferredembodiment, the hybridization conditions comprise 2×SSPE and 63 degreesCelsius for both the hybridization and washing steps.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprises a substitution mutation at a positionequivalent or analogous to a) amino acid residue 48, 51, 52, 54, 92, 95,96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, or 238 of SEQ IDNO: 1, b) amino acid residues 96 and 119 of SEQ ID NO: 1, c) amino acidresidues 110 and 128 of SEQ ID NO: 1, d) amino acid residues 52 and 110of SEQ ID NO: 1, e) amino acid residues 107, 110, and 127 of SEQ ID NO:1, or f) amino acid residues 52, 107 and 127 of SEQ ID NO: 1. In anotherembodiment, the Group H nuclear receptor ligand binding domain comprisessubstitution mutations that results in substitution mutation at aposition equivalent or analogous to amino acid residues 107 and 127 andinsertion of amino acid residue 259 of SEQ ID NO: 1. In a preferredembodiment, the Group H nuclear receptor ligand binding domain is froman ecdysone receptor.

Preferably, the Group H nuclear receptor ligand binding domain comprisesa substitution of a) an asparagine, arginine, tyrosine, tryptophan,leucine or lysine residue at a position equivalent to analogous to aminoacid residue 48 of SEQ ID NO: 1, b) a methionine, asparagine or leucineresidue at a position equivalent or analogous to amino acid residue 51of SEQ ID NO: 1, c) a leucine, proline, methionine, arginine,tryptophan, glycine, glutamine or glutamic acid residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, d) atryptophan or threonine residue at a position equivalent or analogous toamino acid 54 of SEQ ID NO: 1, e) a leucine or glutamic acid residue ata position equivalent or analogous to amino acid 92 of SEQ ID NO: 1, f)a histidine, methionine or tryptophan residue at a position equivalentor analogous to amino acid residue 95 of SEQ ID NO: 1, g) a leucine,serine, glutamic acid or tryptophan residue at a position equivalent oranalogous to amino acid residue 96 of SEQ ID NO: 1, h) a tryptophan,proline, leucine, methionine or asparagine at a position equivalent oranalogous to amino acid 109 of SEQ ID NO: 1, i) a glutamic acid,tryptophan or asparagine residue at a position equivalent or analogousto amino acid residue 110 of SEQ ID NO: 1, j) a phenylalanine residue ata position equivalent or analogous to amino acid 119 of SEQ ID NO: 1, k)a tryptophan or methionine residue at a position equivalent or analogousto amino acid 120 of SEQ ID NO: 1, 1) a glutamic acid, proline, leucine,cysteine, tryptophan, glycine, isoleucine, asparagine, serine, valine orarginine residue at a position equivalent or analogous to amino acid 125of SEQ ID NO: 1, m) a phenylalanine residue at a position equivalent oranalogous to amino acid 128 of SEQ ID NO: 1, n) a methionine,asparagine, glutamic acid or valine residue at a position equivalent oranalogous to amino acid 132 of SEQ ID NO: 1, o) an alanine, lysine,tryptophan or tyrosine residue at a position equivalent or analogous toamino acid residue 219 of SEQ ID NO: 1, p) a lysine, arginine ortyrosine residue at a position equivalent or analogous to amino acidresidue 223 of SEQ ID NO: 1, q) a methionine, arginine, tryptophan orisoleucine residue at a position equivalent or analogous to amino acid234 of SEQ ID NO: 1, r) a proline, glutamic acid, leucine, methionine ortyrosine residue at a position equivalent or analogous to amino acid 238of SEQ ID NO: 1, s) a phenylalanine residue at a position equivalent oranalogous to amino acid 119 of SEQ ID NO: 1 and a threonine residue at aposition equivalent or analogous to amino acid 96 of SEQ ID NO: 1, t) aproline residue at a position equivalent or analogous to amino acid 110of SEQ ID NO: 1 and a phenylalanine residue at a position equivalent oranalogous to amino acid 128 of SEQ ID NO: 1, u) a valine residue at aposition equivalent or analogous to amino acid 52 of SEQ ID NO: 1 and aproline residue at a position equivalent or analogous to amino acid 110of SEQ ID NO: 1, v) an isoleucine residue at a position equivalent oranalogous to amino acid 107 of SEQ ID NO: 1, a glutamic acid residue ata position equivalent or analogous to amino acid 127 of SEQ ID NO: 1 anda proline residue at a position equivalent or analogous to amino acid110 of SEQ ID NO: 1, or w) an isoleucine residue at a positionequivalent or analogous to amino acid 107 of SEQ ID NO: 1, a glutamicacid residue at a position equivalent or analogous to amino acid 127 ofSEQ ID NO: 1 and a valine residue at a position equivalent or analogousto amino acid 52 of SEQ ID NO: 1. In another embodiment, the Group Hnuclear receptor ligand binding domain comprises a substitution of anisoleucine residue at a position equivalent or analogous to amino acid107 of SEQ ID NO: 1, a glutamic acid residue at a position equivalent oranalogous to amino acid 127 of SEQ ID NO: 1 and insertion of a glycineresidue at a position equivalent or analogous to amino acid 259 of SEQID NO: 1. In a preferred embodiment, the Group H nuclear receptor ligandbinding domain is from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain polypeptide comprising a substitutionmutation, wherein the substitution mutation is selected from the groupconsisting of F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, I51L,T52M, T52V, T52L, T52E, T52P, T52R, T52W, T52G, T52Q, M54W, M54T, M92L,M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, F109W, F109P, F109L,F109M, F109N, A110E, A110N, A110W, N119F, Y120W, Y120M, M125P, M125R,M125E, M125L, M125C, M125W, M125G, M1251, M125N, M125S, M125V, V128F,L132M, L132N, L132V, L132E, M219K, M219W, M219Y, M219A, L223K, L223R,L223Y, L234M, L2341, L234R, L234W, W238P, W238E, W238Y, W238M, W238L,N119F/V96T, T52V/A110P, V128F/A110P, V107I/Y127E/T52V, andV107I/Y127E/A110P substitution mutation of SEQ ID NO: 1. In anotherspecific embodiment, the Group H nuclear receptor ligand binding domaincomprising a substitution mutation is an ecdysone receptor ligandbinding domain polypeptide comprising substitution mutation V107I/Y127Eof SEQ ID NO: 1, which further comprises insertion mutation G259 of SEQID NO: 1 (V107I/Y127E/G259).

The DNA binding domain can be any DNA binding domain with a knownresponse element, including synthetic and chimeric DNA binding domains,or analogs, combinations, or modifications thereof. Preferably, the DBDis a GAL4 DBD, a LexA DBD, a transcription factor DBD, a Group H nuclearreceptor member DBD, a steroid/thyroid hormone nuclear receptorsuperfamily member DBD, or a bacterial LacZ DBD. More preferably, theDBD is an EcR DBD [SEQ ID NO: 4 (polynucleotide) or SEQ ID NO: 5(polypeptide)], a GAL4 DBD [SEQ ID NO: 6 (polynucleotide) or SEQ ID NO:7 (polypeptide)], or a LexA DBD [(SEQ ID NO: 8 (polynucleotide) or SEQID NO: 9 (polypeptide)].

The transactivation domain (abbreviated “AD” or “TA”) may be any Group Hnuclear receptor member AD, steroid/thyroid hormone nuclear receptor AD,synthetic or chimeric AD, polyglutamine AD, basic or acidic amino acidAD, a VP16 AD, a GAL4 AD, an NF-KB AD, a BP64 AD, a B42 acidicactivation domain (B42AD), a p65 transactivation domain (p65AD), or ananalog, combination, or modification thereof. In a specific embodiment,the AD is a synthetic or chimeric AD, or is obtained from an EcR, aglucocorticoid receptor, VP16, GAL4, NF-kB, or B42 acidic activationdomain AD. Preferably, the AD is an EcR AD [SEQ ID NO: 10(polynucleotide) or SEQ ID NO: 11 (polypeptide)], a VP16 AD [SEQ ID NO:12 (polynucleotide) or SEQ ID NO: 13 (polypeptide)], a B42 AD [SEQ IDNO: 14 (polynucleotide) or SEQ ID NO: 15 (polypeptide)], or a p65 AD[SEQ ID NO: 16 (polynucleotide) or SEQ ID NO: 17 (polypeptide)].

In a specific embodiment, the gene expression cassette encodes a hybridpolypeptide comprising either a) a DNA-binding domain encoded by apolynucleotide comprising a nucleic acid sequence of SEQ ID NO: 4, SEQID NO: 6, or SEQ ID NO: 8, or b) a transactivation domain encoded by apolynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; and a Group H nuclearreceptor ligand binding domain comprising a substitution mutationencoded by a polynucleotide according to the invention. Preferably, theGroup H nuclear receptor ligand binding domain comprising a substitutionmutation is an ecdysone receptor ligand binding domain comprising asubstitution mutation encoded by a polynucleotide according to theinvention.

In another specific embodiment, the gene expression cassette encodes ahybrid polypeptide comprising either a) a DNA-binding domain comprisingan amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,or b) a transactivation domain comprising an amino acid sequence of SEQID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17; and a Group Hnuclear receptor ligand binding domain comprising a substitutionmutation according to the invention. Preferably, the Group H nuclearreceptor ligand binding domain comprising a substitution mutation is anecdysone receptor ligand binding domain comprising a substitutionmutation according to the invention.

The present invention also provides a gene expression cassettecomprising: i) a response element comprising a domain recognized by apolypeptide comprising a DNA binding domain; ii) a promoter that isactivated by a polypeptide comprising a transactivation domain; and iii)a gene whose expression is to be modulated.

The response element (“RE”) may be any response element with a known DNAbinding domain, or an analog, combination, or modification thereof. Asingle RE may be employed or multiple REs, either multiple copies of thesame RE or two or more different REs, may be used in the presentinvention. In a specific embodiment, the RE is an RE from GAL4(“GAL4RE”), LexA, a Group H nuclear receptor RE, a steroid/thyroidhormone nuclear receptor RE, or a synthetic RE that recognizes asynthetic DNA binding domain. Preferably, the RE is an ecdysone responseelement (EcRE) comprising a polynucleotide sequence of SEQ ID NO: 18, aGAL4RE comprising a polynucleotide sequence of SEQ ID NO: 19, or a LexARE (operon, “op”) comprising a polynucleotide sequence of SEQ ID NO: 20(“2XLexAopRE”).

A steroid/thyroid hormone nuclear receptor DNA binding domain,activation domain or response element according to the invention may beobtained from a steroid/thyroid hormone nuclear receptor selected fromthe group consisting of thyroid hormone receptor a (TRα), thyroidreceptor 1 (c-erbA-1), thyroid hormone receptor β (TRβ), retinoic acidreceptor α (RARα), retinoic acid receptor β (RARβ, HAP), retinoic acidreceptor γ (RARγ), retinoic acid receptor gamma-like (RARD), peroxisomeproliferator-activated receptor α (PPARα), peroxisomeproliferator-activated receptor β (PPARβ), peroxisomeproliferator-activated receptor δ (PPARδ, NUC-1), peroxisomeproliferator-activator related receptor (FFAR), peroxisomeproliferator-activated receptor γ (PPARγ), orphan receptor encoded bynon-encoding strand of thyroid hormone receptor α (REVERBα), v-erb Arelated receptor (EAR-1), v-erb related receptor (EAR-1A), γ), orphanreceptor encoded by non-encoding strand of thyroid hormone receptor β(REVERBβ), v-erb related receptor (EAR-1β), orphan nuclear receptor BD73(BD73), rev-erbA-related receptor (RVR), zinc finger protein 126 (HZF2),ecdysone-inducible protein E75 (E75), ecdysone-inducible protein E78(E78), Drosophila receptor 78 (DR-78), retinoid-related orphan receptorα (RORα), retinoid Z receptor α (RZRα), retinoid related orphan receptorβ (RORβ), retinoid Z receptor β (RZRβ), retinoid-related orphan receptorγ (RORγ), retinoid Z receptor γ (RZRγ), retinoid-related orphan receptor(TOR), hormone receptor 3 (HR-3), Drosophila hormone receptor 3 (DHR-3),Manduca hormone receptor (MHR-3), Galleria hormone receptor 3 (GHR-3),C. elegans nuclear receptor 3 (CNR-3), Choristoneura hormone receptor 3(CHR-3), C. elegans nuclear receptor 14 (CNR-14), ecdysone receptor(ECR), ubiquitous receptor (UR), orphan nuclear receptor (OR-1), NER-1,receptor-interacting protein 15 (RIP-15), liver X receptor β (LXRβ),steroid hormone receptor like protein (RLD-1), liver X receptor (LXR),liver X receptor α (LXRα), farnesoid X receptor (FXR),receptor-interacting protein 14 (RIP-14), HRR-1, vitamin D receptor(VDR), orphan nuclear receptor (ONR-1), pregnane X receptor (PXR),steroid and xenobiotic receptor (SXR), benzoate X receptor (BXR),nuclear receptor (MB-67), constitutive androstane receptor 1 (CAR-1),constitutive androstane receptor α (CARα), constitutive androstanereceptor 2 (CAR-2), constitutive androstane receptor β (CARβ),Drosophila hormone receptor 96 (DHR-96), nuclear hormone receptor 1(NHR-1), hepatocyte nuclear factor 4 (HNF4), hepatocyte nuclear factor4G (HNF4G), hepatocyte nuclear factor 4B (HNF-4B), hepatocyte nuclearfactor 4D (HNF4D, DHNF4), retinoid X receptor a (RXRα), retinoid Xreceptor β (RXRβ), H-2 region II binding protein (H-2RIIBP), nuclearreceptor co-regulator-1 (RCoR-1), retinoid X receptor γ (RXRγ),Ultraspiracle (USP), 2C1 nuclear receptor, chorion factor 1 (CF-1),testicular receptor 2 (TR-2), testicular receptor 2-11 (TR2-11),testicular receptor 4 (TR4), TAK-1, Drosophila hormone receptor (DHR78),Tailless (TLL), tailless homolog (TLX), XTLL, chicken ovalbumin upstreampromoter transcription factor I (COUP-TFI), chicken ovalbumin upstreampromoter transcription factor A (COUP-TFA), EAR-3, SVP-44, chickenovalbumin upstream promoter transcription factor II (COUP-TFII), chickenovalbumin upstream promoter transcription factor B (COUP-TFB), ARP-1,SVP-40, SVP, chicken ovalbumin upstream promoter transcription factor111 (COUP-TFIII), chicken ovalbumin upstream promoter transcriptionfactor G (COUP-TFG), SVP46, EAR-2, estrogen receptor α (ERα), estrogenreceptor β (ERβ), estrogen related receptor 1 (ERR1), estrogen relatedreceptor α (ERRα), estrogen related receptor 2 (ERR2), estrogen relatedreceptor β (ERRβ), glucocorticoid receptor (GR), mineralocorticoidreceptor (MR), progesterone receptor (PR), androgen receptor (AR), nervegrowth factor induced gene B (NGFI-B), nuclear receptor similar toNur-77 (TRS), N10, Orphan receptor (NUR-77), Human early response gene(NAK-1), Nurr related factor 1 (NURR-1), a human immediate-earlyresponse gene (NOT), regenerating liver nuclear receptor 1 (RNR-1),hematopoietic zinc finger 3 (HZF-3), Nur rekated protein −1 (TINOR),Nuclear orphan receptor 1 (NOR-1), NOR1 related receptor (MINOR),Drosophila hormone receptor 38 (DHR-38), C. elegans nuclear receptor 8(CNR-8), C48D5, steroidogenic factor 1 (SF1), endozepine-like peptide(ELP), fushi tarazu factor 1 (FTZ-F1), adrenal 4 binding protein(AD4BP), liver receptor homolog (LRH-1), Ftz-F1-related orphan receptorA (xFFrA), Ftz-F1-related orphan receptor B (xFFrB), nuclear receptorrelated to LRH-1 (FFLR), nuclear receptor related to LRH-1 (PHR),fetoprotein transcriptin factor (FTF), germ cell nuclear factor (GCNFM),retinoid receptor-related testis-associated receptor (RTR), knirps(KNI), knirps related (KNRL), Embryonic gonad (EGON), Drosophila genefor ligand dependent nuclear receptor (EAGLE), nuclear receptor similarto trithorax (ODR7), Trithorax, dosage sensitive sex reversal adrenalhypoplasia congenita critical region chromosome X gene (DAX-1), adrenalhypoplasia congenita and hypogonadotropic hypogonadism (AHCH), and shortheterodimer partner (SHP).

For purposes of this invention, nuclear receptors and Group H nuclearreceptors also include synthetic and chimeric nuclear receptors andGroup H nuclear receptors and their homologs.

Genes of interest for use in Applicants' gene expression cassettes maybe endogenous genes or heterologous genes. Nucleic acid or amino acidsequence information for a desired gene or protein can be located in oneof many public access databases, for example, GENBANK, EMBL, Swiss-Prot,and PIR, or in many biology-related journal publications. Thus, thoseskilled in the art have access to nucleic acid sequence information forvirtually all known genes. Such information can then be used toconstruct the desired constructs for the insertion of the gene ofinterest within the gene expression cassettes used in Applicants'methods described herein.

Examples of genes of interest for use in Applicants' gene expressioncassettes include, but are not limited to: genes encodingtherapeutically desirable polypeptides or products that may be used totreat a condition, a disease, a disorder, a dysfunction, a geneticdefect, such as monoclonal antibodies, enzymes, proteases, cytokines,interferons, insulin, erythropoietin, clotting factors, other bloodfactors or components, viral vectors for gene therapy, virus forvaccines, targets for drug discovery, functional genomics, andproteomics analyses and applications, and the like.

Polynucleotides of the Invention

The novel nuclear receptor-based inducible gene expression system of theinvention comprises at least one gene expression cassette comprising apolynucleotide that encodes a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation. These gene expressioncassettes, the polynucleotides they comprise, and the polypeptides theyencode are useful as components of a nuclear receptor-based geneexpression system to modulate the expression of a gene within a hostcell.

Thus, the present invention provides an isolated polynucleotide thatencodes a Group H nuclear receptor ligand binding domain comprising asubstitution mutation.

In a specific embodiment, the Group H nuclear receptor ligand bindingdomain is encoded by a polynucleotide comprising a codon mutation thatresults in a substitution of an amino acid residue at a positionequivalent or analogous to a) amino acid residue 48, 51, 52, 54, 92, 95,96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, or 238 of SEQ IDNO: 1, b) amino acid residues 96 and 119 of SEQ ID NO: 1, c) amino acidresidues 110 and 128 of SEQ ID NO: 1, d) amino acid residues 52 and 110of SEQ ID NO: 1, e) amino acid residues 107, 110, and 127 of SEQ ID NO:1, or f) amino acid residues 52, 107 and 127 of SEQ ID NO: 1. In anotherembodiment, the Group H nuclear receptor ligand binding domain isencoded by a polynucleotide comprising codon mutations that results insubstitution of amino acid residues at positions equivalent or analogousto amino acid residues 107 and 127, and insertion of amino acid 259 ofSEQ ID NO: 1. In a preferred embodiment, the Group H nuclear receptorligand binding domain is from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain is encoded by a polynucleotide comprising a codonmutation that results in a substitution of a) an asparagine, arginine,tyrosine, tryptophan, leucine or lysine residue at a position equivalentto analogous to amino acid residue 48 of SEQ ID NO: 1, b) a methionine,asparagines or leucine residue at a position equivalent or analogous toamino acid residue 51 of SEQ ID NO: 1, c) a leucine, proline,methionine, arginine, tryptophan, glycine, glutamine or glutamic acidresidue at a position equivalent or analogous to amino acid residue 52of SEQ ID NO: 1, d) a tryptophan or threonine at a position equivalentor analogous to amino acid 54 of SEQ ID NO: 1, e) a leucine or glutamicacid at a position equivalent or analogous to amino acid 92 of SEQ IDNO: 1, f) a histidine, methionine or tryptophan residue at a positionequivalent or analogous to amino acid residue 95 of SEQ ID NO: 1, g) aleucine, serine, glutamic acid or tryptophan residue at a positionequivalent or analogous to amino acid residue 96 of SEQ ID NO: 1, h) atryptophan, proline, leucine, methionine or asparagine at a positionequivalent or analogous to amino acid 109 of SEQ ID NO: 1, i) a glutamicacid, tryptophan or asparagine residue at a position equivalent oranalogous to amino acid residue 110 of SEQ ID NO: 1, j) a phenylalanineat a position equivalent or analogous to amino acid 119 of SEQ ID NO: 1,k) a tryptophan or methionine at a position equivalent or analogous toamino acid 120 of SEQ ID NO: 1, 1) a glutamic acid, proline, leucine,cysteine, tryptophan, glycine, isoleucine, asparagine, serine, valine orarginine at a position equivalent or analogous to amino acid 125 of SEQID NO: 1, m) a phenylalanine at a position equivalent or analogous toamino acid 128 of SEQ ID NO: 1, n) a methionine, asparagine, glutamicacid or valine at a position equivalent or analogous to amino acid 132of SEQ ID NO: 1, o) an alanine, lysine, tryptophan or tyrosine residueat a position equivalent or analogous to amino acid residue 219 of SEQID NO: 1, p) a lysine, arginine or tyrosine residue at a positionequivalent or analogous to amino acid residue 223 of SEQ ID NO: 1, q) amethionine, arginine, tryptophan or isoleucine at a position equivalentor analogous to amino acid 234 of SEQ ID NO: 1, r) a proline, glutanicacid, leucine, methionine or tyrosine at a position equivalent oranalogous to amino acid 238 of SEQ ID NO: 1, s) a phenylalanine at aposition equivalent or analogous to amino acid 119 of SEQ ID NO: 1 and athreonine at a position equivalent or analogous to amino acid 96 of SEQID NO: 1, t) a proline at a position equivalent or analogous to aminoacid 110 of SEQ ID NO: 1 and a phenylalanine at a position equivalent oranalogous to amino acid 128 of SEQ ID NO: 1, u) a valine residue at aposition equivalent or analogous to amino acid 52 of SEQ ID NO: 1 and aproline residue at a position equivalent or analogous to amino acid 110of SEQ ID NO: 1, v) an isoleucine at a position equivalent or analogousto amino acid 107 of SEQ ID NO: 1, a glutamic acid at a positionequivalent or analogous to amino acid 127 of SEQ ID NO: 1 and a prolineat a position equivalent or analogous to amino acid 110 of SEQ ID NO: 1,or w) an isoleucine at a position equivalent or analogous to amino acid107 of SEQ ID NO: 1, a glutamic acid at a position equivalent oranalogous to amino acid 127 of SEQ ID NO: 1 and a valine at a positionequivalent or analogous to amino acid 52 of SEQ ID NO: 1. In anotherembodiment, the Group H nuclear receptor ligand binding domain isencoded by a polynucleotide comprising codon mutations that results insubstitution of an isoleucine residue at a position equivalent oranalogous to amino acid 107 of SEQ ID NO: 1, a glutamic acid residue ata position equivalent or analogous to amino acid 127 of SEQ ID NO: 1 andinsertion of a glycine residue at a position equivalent or analogous toamino acid 259 of SEQ ID NO: 1. In a preferred embodiment, the Group Hnuclear receptor ligand binding domain is from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain comprising a substitution mutationencoded by a polynucleotide comprising a codon mutation that results ina substitution mutation selected from the group consisting of F48Y,F48W, F48L, F48N, F48R, F48K, I51M, I51N, I51L, T52M, T52V, T52L, T52E,T52P, T52R, T52W, T52G, T52Q, M54W, M54T, M92L, M92E, R95H, R95M, R95W,V96L, V96W, V96S, V96E, F109W, F109P, F109L, F109M, F109N, A110E, A110N,A110W, N119F, Y120W, Y120M, M125P, M125R, M125E, M125L, M125C, M125W,M125G, M1251, M125N, M125S, M125V, V128F, L132M, L132N, L132V, L132E,M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L2341, L234R,L234W, W238P, W238E, W238Y, W238M, W238L, N119F/V96T, V128F/A110P,T52V/A110P, V107V/Y127E/T52V, and V107I/Y127E/A110P substitutionmutation of SEQ ID NO: 1. In another embodiment, the Group H nuclearreceptor ligand binding domain comprising a substitution mutation is anecdysone receptor ligand binding domain comprising a substitutionmutation encoded by a polynucleotide comprising a codon mutation thatresults in substitution mutation V107I/Y127E of SEQ ID NO: 1, whichfurther comprises insertion mutation G259 of SEQ ID NO: 1(V1071I/Y27E/G259).

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain comprising a substitution mutationencoded by a polynucleotide that hybridizes to a polynucleotidecomprising a codon mutation that results in a substitution mutationselected from the group consisting of F48Y, F48W, F48L, F48N, F48R,F48K, 151M, 151N, 151L, T52M, T52V, T52L, T52E, T52P, T52R, T52W, T52G,T52Q, M54W, M54T, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E,F109W, F109P, F109L, F109M, F109N, A110E, A110N, A110W, N119F, Y120W,Y120M, M125P, M125R, M125E, M125L, M125C, M125W, M125G, M125I, M125N,M125S, M125V, V128F, L132M, L132N, L132V, L132E, M219K, M219W, M219Y,M219A, L223K, L223R, L223Y, L234M, L234I, L234R, L234W, W238P, W238E,W238Y, W238M, W238L, N119F/V96T, V128F/A110P, T52V/A110P,V107I/Y127E/T52V, and V107I/Y127E/A110P of SEQ ID NO: 1 underhybridization conditions comprising a hybridization step in less than500 mM salt and at least 37 degrees Celsius, and a washing step in2×SSPE at least 63 degrees Celsius. In a preferred embodiment, thehybridization conditions comprise less than 200 mM salt and at least 37degrees Celsius for the hybridization step. In another preferredembodiment, the hybridization conditions comprise 2×SSPE and 63 degreesCelsius for both the hybridization and washing steps.

The present invention also provides an isolated polynucleotide thatencodes a polypeptide selected from the group consisting of a) apolypeptide comprising a transactivation domain, a DNA-binding domain,and a Group H nuclear receptor ligand binding domain comprising asubstitution mutation according to the invention; b) a polypeptidecomprising a DNA-binding domain and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation according to theinvention; and c) a polypeptide comprising a transactivation domain anda Group H nuclear receptor ligand binding domain comprising asubstitution mutation according to the invention.

In a specific embodiment, the isolated polynucleotide encodes a hybridpolypeptide selected from the group consisting of a) a hybridpolypeptide comprising a transactivation domain, a DNA-binding domain,and a Group H nuclear receptor ligand binding domain comprising asubstitution mutation according to the invention; b) a hybridpolypeptide comprising a DNA-binding domain and a Group H nuclearreceptor ligand binding domain comprising a substitution mutationaccording to the invention; and c) a hybrid polypeptide comprising atransactivation domain and a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation according to the invention.

The present invention also relates to an isolated polynucleotideencoding a Group H nuclear receptor ligand binding domain comprising asubstitution mutation, wherein the substitution mutation affects ligandbinding activity or ligand sensitivity of the Group H nuclear receptorligand binding domain.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation reduces non-ecdysteroid diacylhydrazine bindingactivity or non-ecdysteroid diacylhydrazine sensitivity of the Group Hnuclear receptor ligand binding domain. Preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionof an amino acid residue at a position equivalent or analogous to aminoacid residue 48, 51, 52, 54, 92, 95, 96, 109, 120, 125, 219, 223, 234 or238 of SEQ ID NO: 1. More preferably, the isolated polynucleotidecomprises a codon mutation that results in a substitution of a) anasparagine residue at a position equivalent or analogous to amino acidresidue 48 or 109 of SEQ ID NO: 1, b) a leucine residue at a positionequivalent or analogous to amino acid residue 51, 92, 96 or 238 of SEQID NO: 1, c) a glutamic acid residue at a position equivalent oranalogous to amino acid residue 52, 92, 96, 125 or 238 of SEQ ID NO: 1,d) a tryptophan residue at a position equivalent or analogous to aminoacid residue 54, 95, 96, 120, 219 or 234 of SEQ ID NO: 1, e) amethionine residue at a position equivalent or analogous to amino acidresidue 51, 52, 120, 234 or 238 of SEQ ID NO: 1, f) an alanine residueat a position equivalent or analogous to amino acid residue 219 of SEQID NO: 1, g) a lysine residue at a position equivalent or analogous toamino acid residue 48, 219 or 223 of SEQ ID NO: 1, h) an isoleucine,arginine or tryptophan residue at a position equivalent or analogous toamino acid residue 234 of SEQ ID NO: 1, i) a tyrosine residue at aposition equivalent or analogous to amino acid residue 219 or 238 of SEQID NO: 1, j) a valine residue at a position equivalent or analogous toamino acid residue 125 of SEQ ID NO: 1, k) a glycine or glutamineresidue at a position equivalent or analogous to amino acid residue 52of SEQ ID NO: 1 or 1) an arginine residue at a position equivalent oranalogous to amino acid residue 52 or 223 of SEQ ID NO: 1. Even morepreferably, the isolated polynucleotide comprises a codon mutation thatresults in a substitution mutation of F48N, F48K, I51L, I51M, T52E,T52M, T52R, T52G, T52Q, M54W, M92L, M92E, R95W, V96W, V96E, V96L, F109N,Y120M, Y120W, M125E, M125V, M219A, M219K, M219W, M219Y, L223K, L223R,L234M, L2341, L234R, L234W, W238E, W238Y, W238L or W238M of SEQ ID NO:1.

In addition, the present invention also relates to an isolatedpolynucleotide encoding a Group H nuclear receptor ligand binding domaincomprising a substitution mutation, wherein the substitution mutationenhances ligand binding activity or ligand sensitivity of the Group Hnuclear receptor ligand binding domain.

In a specific embodiment, the present invention relates to an isolatedpolynucleotide encoding a Group H nuclear receptor ligand binding domaincomprising a substitution mutation, wherein the substitution mutationgenerally enhances ecdysteroid binding activity or ecdysteroidsensitivity of the Group H nuclear receptor ligand binding domain.Preferably, the isolated polynucleotide comprises a codon mutation thatresults in a substitution of an amino acid residue at a positionequivalent or analogous to a) amino acid residue 96 of SEQ ID NO: 1 orb) amino acid residues 96 and 119 of SEQ ID NO: 1. More preferably, theisolated polynucleotide comprises a codon mutation that results in asubstitution of a) a serine residue at a position equivalent oranalogous to amino acid residue 96 of SEQ ID NO: 1 or b) a threonineresidue at a position equivalent or analogous to amino acid residue 96of SEQ ID NO: 1 and a phenylalanine residue at a position equivalent oranalogous to amino acid residue 119 of SEQ ID NO: 1. Even morepreferably, the isolated polynucleotide comprises a codon mutation thatresults in a substitution mutation of V96T or N119F/V96T of SEQ ID NO:1.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation generally enhances non-ecdysteroid diacylhydrazinebinding activity or non-ecdysteroid diacylhydrazine sensitivity of theGroup H nuclear receptor ligand binding domain. Preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionof an amino acid residue at a position equivalent or analogous to a)amino acid residue 48, 52, 54, 109, 110, 125, 132 or 223 of SEQ ID NO: 1or b) amino acid residues 52 and 110 of SEQ ID NO: 1. More preferably,the isolated polynucleotide comprises a codon mutation that results in asubstitution of a) a tyrosine, tryptophan, arginine or leucine residueat a position equivalent or analogous to amino acid residue 48 of SEQ IDNO: 1, b) a leucine residue at a position equivalent or analogous toamino acid residue 52 of SEQ ID NO: 1, c) a threonine residue at aposition equivalent or analogous to amino acid residue 54 of SEQ ID NO:1, d) methionine residue at a position equivalent or analogous to aminoacid residue 109 of SEQ ID NO: 1, e) a proline, glutamic acid orasparagine residue at a position equivalent or analogous to amino acidresidue 110 of SEQ ID NO: 1, f) an isoleucine, asparagine or glycineresidue at a position equivalent or analogous to amino acid residue 125of SEQ ID NO: 1, g) a glutamic acid residue at a position equivalent oranalogous to amino acid residue 132 of SEQ ID NO: 1, h) a tyrosineresidue at a position equivalent or analogous to amino acid residue 223of SEQ ID NO: 1 or i) a valine residue at a position equivalent oranalogous to amino acid 52 of SEQ ID NO: 1 and a proline residue residueat a position equivalent or analogous to amino acid 110 of SEQ ID NO: 1.Even more preferably, the isolated polynucleotide comprises a codonmutation that results in a substitution mutation of F48Y, F48W, F48L,F48R, T52L, M54T, F109M, A110P, A110E, A110N, M1251, M125G, M125N,L132E, L223Y or T52V/A110P of SEQ ID NO: 1.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation generally enhances non-ecdysteroid diacylhydrazineand non-ecdysteroid tetrahydroquinoline binding activity ornon-ecdysteroid diacylhydrazine and non-ecdysteroid tetrahydroquinolinesensitivity of the Group H nuclear receptor ligand binding domain.Preferably, the isolated polynucleotide comprises a codon mutation thatresults in a substitution of a) amino acid residues at a positionequivalent or analogous to amino acid residues 107 and 127 of SEQ ID NO:1 or b) amino acid residues 107, 110 and 127 of SEQ ID NO: 1. Morepreferably, the isolated polynucleotide comprises a codon mutation thatresults in a substitution of a) an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamic acid residue at a position equivalent or analogous to aminoacid residue 127 of SEQ ID NO: 1 or b) an isoleucine residue at aposition equivalent or analogous to amino acid residue 107 of SEQ ID NO:1, a proline residue at a position equivalent or analogous to amino acidresidue 110 of SEQ ID NO: 1 and a glutamic acid residue at a positionequivalent or analogous to amino acid residue 127 of SEQ ID NO: 1. Evenmore preferably, the isolated polynucleotide comprises a codon mutationthat results in a substitution mutation of V107I/Y127E orV107I/Y127E/A110P of SEQ ID NO: 1.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation generally enhances both ecdysteroid bindingactivity or ecdysteroid sensitivity and non-ecdysteroid diacylhydrazinebinding activity or non-ecdysteroid diacylhydrazine sensitivity of theGroup H ligand binding domain. Preferably, the isolated polynucleotidecomprises a codon mutation that results in a substitution of an aminoacid residue at a position equivalent or analogous to a) amino acidresidue 109, 132 or W238P of SEQ ID NO: 1, b) amino acid residues 52,107 and 127 of SEQ ID NO: 1 or c) amino acid residues 107 and 127 of SEQID NO: 1, and insertion of amino acid 259 of SEQ ID NO: 1. Morepreferably, the isolated polynucleotide comprises a codon mutation thatresults in a substitution of a) tryptophan residue at a positionequivalent or analogous to amino acid residue 109 of SEQ ID NO: 1, b) avaline or methionine residue at a position equivalent or analogous toamino acid residue 132 of SEQ ID NO: 1, c) a proline residue at aposition equivalent or analogous to amino acid residue 238 of SEQ ID NO:1, d) an isoleucine residue at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 1, a glutamic acid residue at aposition equivalent or analogous to amino acid residue 127 of SEQ ID NO:1 and a valine residue at a position equivalent or analogous to aminoacid residue 132 of SEQ ID NO: 1 or e) an isoleucine residue at aposition equivalent or analogous to amino acid 107 of SEQ ID NO: 1, aglutamic acid residue at a position equivalent or analogous to aminoacid 127 of SEQ ID NO: 1 and insertion of a glycine residue at aposition equivalent or analogous to amino acid 259 of SEQ ID NO: 1. Evenmore preferably, the isolated polynucleotide comprises a codon mutationthat results in a substitution mutation of F109W, L132M, L132V, W238P,V107I/Y127E/T52V or V1071/Y127E/259G of SEQ ID NO: 1. In anotherembodiment, the isolated polynucleotide comprises a codon mutation thatresults in substitution mutation V107I/Y127E of SEQ ID NO: 1 furthercomprising insertion mutation G259 of SEQ ID NO: 1 (V1071/Y127E/G259).

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation generally enhances non-ecdysteroidtetrahydroquinoline binding activity or non-ecdysteroidtetrahydroquinoline sensitivity of the Group H nuclear receptor ligandbinding domain. Preferably, the isolated polynucleotide comprises acodon mutation that results in a substitution of a) amino acid residueat a position equivalent or analogous to amino acid residues 110 or 128of SEQ ID NO: 1 or b) amino acid residues at a position equivalent oranalogous to amino acid residues 110 and 128 of SEQ ID NO: 1. Morepreferably, the isolated polynucleotide comprises a codon mutation thatresults in a substitution of a) a tryptophan residue at a positionequivalent or analogous to amino acid residue 110 of SEQ ID NO: 1, b) aphenylalanine residue at a position equivalent or analogous to aminoaicd residue 128 of SEQ ID NO: 1 or c) a proline residue at a positionequivalent or analogous to amino acid residue 110 of SEQ ID NO: 1 and aphenylalanine residue at a position equivalent or analogous to aminoacid residue 128 of SEQ ID NO: 1. Even more preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionmutation A110W, V128F or V128F/A110P of SEQ ID NO: 1.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation differentially responds to non-ecdysteroiddiacylhydrazine ligands. Preferably, the isolated polynucleotidecomprises a codon mutation that results in a substitution of an aminoacid residue at a position equivalent or analogous to amino acidresidues 52, 95, 109, 125 or 132 of SEQ ID NO: 1. More preferably, theisolated polynucleotide comprises a codon mutation that results in asubstitution of a) a proline residue at a position equivalent oranalogous to amino acid residue 52 of SEQ ID NO: 1, b) a histidine ormethionine residue at a position equivalent or analogous to amino acidresidue 95 of SEQ ID NO:1, c) a leucine residue at a position equivalentor analogous to amino acid residue 109 of SEQ ID NO: 1, d) a leucine,tryptophan, arginine, cysteine or proline residue at a positionequivalent or analogous to amino acid residue 125 of SEQ ID NO: 1 or e)a methionine residue at a position equivalent or analogous to amino acidresidue 132 of SEQ ID NO: 1. Even more preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionmutation T52P, T52W, R95H, R95M, F109L, M125L, M125W, M125R, M125C,M125P or L132M of SEQ ID NO: 1.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation differentially responds to non-ecdysteroiddiacylhydrazine ligands. More preferably the isolated polynucleotidecomprises a codon mutation that results in a substitution of a) a lysineor arginine residue at a position equivalent or analogous to amino acidresidue 48 of SEQ ID NO: 1, b) a glycine, glutamine, methionine,arginine or tryptophan residue at a position equivalent or analogous toamino acid residue 52 of SEQ ID NO: 1, c) an isoleucine, glycine,asparagine, serine or valine residue at a position equivalent oranalogous to amino acid residue 125 of SEQ ID NO: 1, d) a glutamic acidresidue at a position equivalent or analogous to amino acid residue 132of SEQ ID NO: 1, e) a lysine, tryptophan or tyrosine residue at aposition equivalent or analogous to amino acid residue 219 of SEQ ID NO:1, f) an arginine or tyrosine residue at a position equivalent oranalogous to amino acid residue 223 of SEQ ID NO: 1 or g) leucine ormethionine residue at a position equivalent or analogous to amino acidresidue 238 of SEQ ID NO: 1. Even more preferably the isolatedpolynucleotide comprises a codon mutation that results in a substitutionmutation F48 K, F48R, T52G, T52Q, T52M, T52R, T52W, M1251, M125G, M125N,M125S, M125V, L132E, M219K, M219W, M219Y, L223R, L223Y, W238L or W238Mof SEQ ID NO: 1.

In addition, the present invention relates to an expression vectorcomprising a polynucleotide according the invention, operatively linkedto a transcription regulatory element. Preferably, the polynucleotideencoding a nuclear receptor ligand binding domain comprising asubstitution mutation is operatively linked with an expression controlsequence permitting expression of the nuclear receptor ligand bindingdomain in an expression competent host cell. The expression controlsequence may comprise a promoter that is functional in the host cell inwhich expression is desired. The vector may be a plasmid DNA molecule ora viral vector. Preferred viral vectors include retrovirus, adenovirus,adeno-associated virus, herpes virus, and vaccinia virus. The inventionfurther relates to a replication defective recombinant virus comprisingin its genome, the polynucleotide encoding a nuclear receptor ligandbinding domain comprising a substitution mutation as described above.Thus, the present invention also relates to an isolated host cellcomprising such an expression vector, wherein the transcriptionregulatory element is operative in the host cell.

The present invention also relates to an isolated polypeptide encoded bya polynucleotide according to the invention.

Polypeptides of the Invention

The novel nuclear receptor-based inducible gene expression system of theinvention comprises at least one gene expression cassette comprising apolynucleotide that encodes a polypeptide comprising a Group H nuclearreceptor ligand binding domain comprising a substitution mutation. Thus,the present invention also provides an isolated polypeptide comprising aGroup H nuclear receptor ligand binding domain comprising a substitutionmutation according to the invention.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprises a substitution mutation at a positionequivalent or analogous to a) amino acid residue 48, 51, 52, 54, 92, 95,96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, or 238 of SEQ IDNO: 1, b) amino acid residues 96 and 119 of SEQ ID NO: 1, c) amino acidresidues 110 and 128 of SEQ ID NO: 1, d) amino acid residues 52 and 110of SEQ ID NO: 1, e) amino acid residues 107, 110, and 127 of SEQ ID NO:1, or f) amino acid residues 52, 107 and 127 of SEQ ID NO: 1. In anotherembodiment, the Group H nuclear receptor ligand binding domain comprisessubstitution mutation at positions equivalent or analogous to amino acidresidues 107 and 127, and insertion of amino acid 259 of SEQ ID NO: 1.In a preferred embodiment, the Group H nuclear receptor ligand bindingdomain is from an ecdysone receptor.

Preferably, the Group H nuclear receptor ligand binding domain comprisesa substitution of a) an asparagine, arginine, tyrosine, tryptophan,leucine or lysine residue at a position equivalent to analogous to aminoacid residue 48 of SEQ ID NO: 1, b) a methionine, asparagines or leucineresidue at a position equivalent or analogous to amino acid residue 51of SEQ ID NO: 1, c) a leucine, proline, methionine, arginine,tryptophan, glycine, glutamine or glutamic acid residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, d) atryptophan or threonine at a position equivalent or analogous to aminoacid 54 of SEQ ID NO: 1, e) a leucine or glutamic acid at a positionequivalent or analogous to amino acid 92 of SEQ ID NO: 1, f) ahistidine, methionine or tryptophan residue at a position equivalent oranalogous to amino acid residue 95 of SEQ ID NO: 1, g) a leucine,serine, glutamic acid or tryptophan residue at a position equivalent oranalogous to amino acid residue 96 of SEQ ID NO: 1, h) a tryptophan,proline, leucine, methionine or asparagine at a position equivalent oranalogous to amino acid 109 of SEQ ID NO: 1, i) a glutamic acid,tryptophan or asparagine residue at a position equivalent or analogousto amino acid residue 110 of SEQ ID NO: 1, j) a phenylalanine at aposition equivalent or analogous to amino acid 119 of SEQ ID NO: 1, k) atryptophan or methionine at a position equivalent or analogous to aminoacid 120 of SEQ ID NO: 1, l) a glutamic acid, proline, leucine,cysteine, tryptophan, glycine, isoleucine, asparagine, serine, valine orarginine at a position equivalent or analogous to amino acid 125 of SEQID NO: 1, m) a phenylalanine at a position equivalent or analogous toamino acid 128 of SEQ ID NO: 1, n) a methionine, asparagine, glutamicacid or valine at a position equivalent or analogous to amino acid 132of SEQ ID NO: 1, o) an alanine, lysine, tryptophan or tyrosine residueat a position equivalent or analogous to amino acid residue 219 of SEQID NO: 1, p) a lysine, arginine or tyrosine residue at a positionequivalent or analogous to amino acid residue 223 of SEQ ID NO: 1, q) amethionine, arginine, tryptophan or isoleucine at a position equivalentor analogous to amino acid 234 of SEQ ID NO: 1, r) a proline, glutamicacid, leucine, methionine or tyrosine at a position equivalent oranalogous to amino acid 238 of SEQ ID NO: 1, s) a phenylalanine at aposition equivalent or analogous to amino acid 119 of SEQ ID NO: 1 and athreonine at a position equivalent or analogous to amino acid 96 of SEQID NO: 1, t) a proline at a position equivalent or analogous to aminoacid 110 of SEQ ID NO: 1 and a phenylalanine at a position equivalent oranalogous to amino acid 128 of SEQ ID NO: 1, u) a valine residue at aposition equivalent or analogous to amino acid 52 of SEQ ID NO: 1 and aproline residue at a position equivalent or analogous to amino acid 110of SEQ ID NO: 1, v) an isoleucine at a position equivalent or analogousto amino acid 107 of SEQ ID NO: 1, a glutamic acid at a positionequivalent or analogous to amino acid 127 of SEQ ID NO: 1 and a prolineat a position equivalent or analogous to amino acid 110 of SEQ ID NO: 1,or w) an isoleucine at a position equivalent or analogous to amino acid107 of SEQ ID NO: 1, a glutamic acid at a position equivalent oranalogous to amino acid 127 of SEQ ID NO: 1 and a valine at a positionequivalent or analogous to amino acid 52 of SEQ ID NO: 1. In anotherembodiment, Group H nuclear receptor ligand binding domain comprises asubstitution of an isoleucine residue at a position equivalent oranalogous to amino acid 107 of SEQ ID NO: 1, a glutamic acid residue ata position equivalent or analogous to amino acid 127 of SEQ ID NO: 1 andinsertion of a glycine residue at a position equivalent or analogous toamino acid 259 of SEQ ID NO: 1. In a preferred embodiment, the Group Hnuclear receptor ligand binding domain is from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain polypeptide comprising a substitutionmutation, wherein the substitution mutation is selected from the groupconsisting of F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, I51L,T52M, T52V, T52L, T52E, T52P, T52R, T52W, T52G, T52Q, M54W, M54T, M92L,M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, F109W, F109P, F109L,F109M, F109N, A110E, A110N, A110W, N119F, Y120W, Y120M, M125P, M125R,M125E, M125L, M125C, M125W, M125G, M1251, M125N, M125S, M125V, V128F,L132M, L132N, L132V, L132E, M219K, M219W, M219Y, M219A, L223K, L223R,L223Y, L234M, L234I, L234R, L234W, W238P, W238E, W238Y, W238M, W238L, N119FN96T, V128F/A110P, T52V/A110P, V107I/Y127E/T52V, andV107I/Y127E/A110P substitution mutation of SEQ ID NO: 1. In anotherembodiment, the Group H nuclear receptor ligand binding domaincomprising a substitution mutation is an ecdysone receptor ligandbinding domain polypeptide comprising a substitution mutation ofV107I/Y127E of SEQ ID NO: 1, which further comprises insertion mutationG259 of SEQ ID NO: 1 (V107I/Y127E/G259).

The present invention also provides an isolated polypeptide selectedfrom the group consisting of a) an isolated polypeptide comprising atransactivation domain, a DNA-binding domain, and a Group H nuclearreceptor ligand binding domain comprising a substitution mutationaccording to the invention; b) an isolated polypeptide comprising aDNA-binding domain and a Group H nuclear receptor ligand binding domaincomprising a substitution mutation according to the invention; and c) anisolated polypeptide comprising a transactivation domain and a Group Hnuclear receptor ligand binding domain comprising a substitutionmutation according to the invention. In a preferred embodiment, theGroup H nuclear receptor ligand binding domain is from an ecdysonereceptor.

The present invention also provides an isolated hybrid polypeptideselected from the group consisting of a) an isolated hybrid polypeptidecomprising a transactivation domain, a DNA-binding domain, and a Group Hnuclear receptor ligand binding domain comprising a substitutionmutation according to the invention; b) an isolated hybrid polypeptidecomprising a DNA-binding domain and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation according to theinvention; and c) an isolated hybrid polypeptide comprising atransactivation domain and a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation according to the invention. Ina preferred embodiment, the Group H nuclear receptor ligand bindingdomain is from an ecdysone receptor.

The present invention also provides an isolated polypeptide comprising aGroup H nuclear receptor ligand binding domain comprising a substitutionmutation that affects ligand binding activity or ligand sensitivity ofthe Group H nuclear receptor ligand binding domain.

In particular, the present invention relates to an isolated Group Hnuclear receptor polypeptide comprising a ligand binding domaincomprising a substitution mutation that reduces ligand binding activityor ligand sensitivity of the Group H nuclear receptor ligand bindingdomain.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation that reducesnon-ecdysteroid diacylhydrazine binding activity or non-ecdysteroiddiacylhydrazine sensitivity of the Group H nuclear receptor ligandbinding domain. Preferably, the isolated polypeptide comprises asubstitution of an amino acid residue at a position equivalent oranalogous to amino acid residue 48, 51, 52, 54, 92, 95, 96, 109, 120,125, 219, 223, 234 or 238 of SEQ ID NO: 1. More preferably, the isolatedpolypeptide comprises a codon mutation that results in a substitution ofa) an asparagine residue at a position equivalent or analogous to aminoacid residue 48 or 109 of SEQ ID NO: 1, b) a leucine residue at aposition equivalent or analogous to amino acid residue 51, 92, 96 or 238of SEQ ID NO: 1, c) a glutamic acid residue at a position equivalent oranalogous to amino acid residue 52, 92, 96, 125 or 238 of SEQ ID NO: 1,d) a tryptophan residue at a position equivalent or analogous to aminoacid residue 54, 95, 96, 120 or 219 of SEQ ID NO: 1, e) a methionineresidue at a position equivalent or analogous to amino acid residue 51,52, 120, 234 or 238 of SEQ ID NO: 1, f) an alanine residue at a positionequivalent or analogous to amino acid residue 219 of SEQ ID NO: 1, g) alysine residue at a position equivalent or analogous to amino acidresidue 48, 219 or 223 of SEQ ID NO: 1, h) an isoleucine, arginine ortryptophan residue at a position equivalent or analogous to amino acidresidue 234 of SEQ ID NO: 1, i) a tyrosine residue at a positionequivalent or analogous to amino acid residue 219 or 238 of SEQ ID NO:1, j) an arginine residue at a position equivalent or analogous to aminoacid residue 52 or 223 of SEQ ID NO: 1, k) a valine residue at aposition equivalent or analogous to amino acid residue 125 of SEQ ID NO:1 or 1) a glycine or glutamine residue at a position equivalent oranalogous to amino acid residue 52 of SEQ ID NO: 1. Even morepreferably, the isolated polypeptide comprises a codon mutation thatresults in a substitution mutation of F48N, I51L, I51M, T52E, T52M,T52R, T52G, T52Q, M54W, M92L, M92E, R95W, V96W, V96E, V96L, F109N,Y120M, Y120W, M125E, M125V, M219A, M219K, M219W, M219Y, L223K, L223R,L234M, L2341, L234W, L234R, W238E, W238L, W238M or W238Y of SEQ ID NO:1.

In addition, the present invention also relates to an isolatedpolypeptide comprising a Group H nuclear receptor ligand binding domaincomprising a substitution mutation that enhances ligand binding activityor ligand sensitivity of the Group H nuclear receptor ligand bindingdomain.

In a specific embodiment, the present invention relates to an isolatedpolypeptide comprising a Group H nuclear receptor ligand binding domaincomprising a substitution mutation that generally enhances ecdysteroidbinding activity or ecdysteroid sensitivity of the Group H nuclearreceptor ligand binding domain. Preferably, the isolated polypeptidecomprises a substitution of an amino acid residue at a positionequivalent or analogous to a) amino acid residue 96 of SEQ ID NO: 1 orb) amino acid residues 96 and 119 of SEQ ID NO: 1. More preferably, theisolated polypeptide comprises a codon mutation that results in asubstitution of a) a serine residue at a position equivalent oranalogous to amino acid residue 96 of SEQ ID NO: 1 or b) a threonineresidue at a position equivalent or analogous to amino acid residue 96of SEQ ID NO: 1 and a phenylalanine residue at a position equivalent oranalogous to amino acid residue 119 of SEQ ID NO: 1. Even morepreferably, the isolated polypeptide comprises a codon mutation thatresults in a substitution mutation of V96T or N119F/V96T of SEQ ID NO:1.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation that generallyenhances diacylhydrazine binding activity or diacylhydrazine sensitivityof the Group H nuclear receptor ligand binding domain. Preferably, theisolated polypeptide comprises a substitution of an amino acid residueat a position equivalent or analogous to a) amino acid residue 48, 52,54, 109, 110, 125, 132 or 223 of SEQ ID NO: 1 or b) amino acid residues52 and 110 of SEQ ID NO: 1. More preferably, the isolated polypeptidecomprises a codon mutation that results in a substitution of a) atyrosine, tryptophan, arginine or leucine residue at a positionequivalent or analogous to amino acid residue 48 of SEQ ID NO: 1, b) aleucine residue at a position equivalent or analogous to amino acidresidue 52 of SEQ ID NO: 1, d) a threonine residue at a positionequivalent or analogous to amino acid residue 54 of SEQ ID NO: 1, e)methionine residue at a position equivalent or analogous to amino acidresidue 109 of SEQ ID NO: 1, f) a proline, glutamic acid or asparagineresidue at a position equivalent or analogous to amino acid residue 110of SEQ ID NO: 1, g) an isoleucine, glycine or asparagine residue at aposition equivalent or analogous to amino acid residue 125 of SEQ ID NO:1, h) a valine residue at a position equivalent or analogous to aminoacid 52 of SEQ ID NO: 1 and a proline residue at a position equivalentor analogous to amino acid 110 of SEQ ID NO: 1, i) a glutamic acidresidue at a position equivalent or analogous to amino acid residue 132of SEQ ID NO: 1 or j) a tyrosine residue at a position equivalent oranalogous to amino acid residue 223 of SEQ ID NO: 1. Even morepreferably, the isolated polypeptide comprises a codon mutation thatresults in a substitution mutation of F48Y, F48W, F48L, F48R, T52L,M54T, F109M, A110P, A110E, A110N, M1251, M125G, M125N, L132E, L223Y orT52V/A110P of SEQ ID NO: 1.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation that generallyenhances both ecdysteroid binding activity or ecdysteroid sensitivityand non-ecdysteroid diacylhydrazine binding activity or non-ecdysteroiddiacylhydrazine sensitivity of the Group H ligand binding domain.Preferably, the isolated polypeptide comprises a substitution of anamino acid residue at a position equivalent or analogous to a) aminoacid residue 109, 132 or W238P of SEQ ID NO: 1, b) amino acid residues52, 107 and 127 of SEQ ID NO: 1 or c) amino acid residues 107 and 127 ofSEQ ID NO: 1, and insertion of amino acid 259 of SEQ ID NO: 1. Morepreferably, the isolated polypeptide comprises a codon mutation thatresults in a substitution of a) tryptophan residue at a positionequivalent or analogous to amino acid residue 109 of SEQ ID NO: 1, b) avaline or methionine residue at a position equivalent or analogous toamino acid residue 132 of SEQ ID NO: 1, c) a proline residue at aposition equivalent or analogous to amino acid residue 238 of SEQ ID NO:1, d) an isoleucine residue at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 1, a glutamic acid residue at aposition equivalent or analogous to amino acid residue 127 of SEQ ID NO:1 and a valine residue at a position equivalent or analogous to aminoacid residue 132 of SEQ ID NO: 1 or e) an isoleucine residue at aposition equivalent or analogous to amino acid 107 of SEQ ID NO: 1, aglutamic acid residue at a position equivalent or analogous to aminoacid 127 of SEQ ID NO: 1 and insertion of a glycine residue at aposition equivalent or analogous to amino acid 259 of SEQ ID NO: 1. Evenmore preferably, the isolated polypeptide comprises a codon mutationthat results in a substitution mutation of F109W, L132M, L132V, W238P orV107I/Y127E/T52V of SEQ ID NO: 1. In another embodiment, the isolatedpolypeptide comprises a codon mutation that results in substitutionmutation V1071/Y127E of SEQ ID NO: 1, which further comprises insertionmutation G259 of SEQ ID NO: 1 (V107I/Y127E/G259).

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation generally enhances diacylhydrazine andtetrahydroquinoline binding activity or diacylhydrazine andtetrahydroquinoline sensitivity of the Group H nuclear receptor ligandbinding domain. Preferably, the isolated polypeptide comprises asubstitution mutation that results in a substitution of a) amino acidresidues at a position equivalent or analogous to amino acid residues107 and 127 of SEQ ID NO: 1 or b) amino acid residues 107, 110 and 127of SEQ ID NO: 1. More preferably, the isolated polypeptide comprises acodon mutation that results in a substitution of a) an isoleucineresidue at a position equivalent or analogous to amino acid residue 107of SEQ ID NO: 1 and a glutamic acid residue at a position equivalent oranalogous to amino acid residue 127 of SEQ ID NO: 1 or b) an isoleucineresidue at a position equivalent or analogous to amino acid residue 107of SEQ ID NO: 1, a proline residue at a position equivalent or analogousto amino acid residue 110 of SEQ ID NO: 1 and a glutamic acid residue ata position equivalent or analogous to amino acid residue 127 of SEQ IDNO: 1. Even more preferably, the isolated polypeptide comprises a codonmutation that results in a substitution mutation of V107I/Y127E orV107I/Y127E/A110P of SEQ ID NO: 1.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation generally enhances non-ecdysteroidtetrahydroquinoline binding activity or non-ecdysteroidtetrahydroquinoline sensitivity of the Group H nuclear receptor ligandbinding domain. Preferably, the isolated polypeptide comprises a codonmutation that results in a substitution of a) amino acid residue at aposition equivalent or analogous to amino acid residues 110 or 128 ofSEQ ID NO: 1 or b) amino acid residues at a position equivalent oranalogous to amino acid residues 110 and 128 of SEQ ID NO: 1. Morepreferably, the isolated polypeptide comprises a codon mutation thatresults in a substitution of a) a tryptophan residue at a positionequivalent or analogous to amino acid residue 110 of SEQ ID NO: 1, b) aphenylalanine residue at a position equivalent or analogous to aminoacid residue 128 of SEQ ID NO: 1 or c) a proline residue at a positionequivalent or analogous to amino acid residue 110 of SEQ ID NO: 1 and aphenylalanine residue at a position equivalent or analogous to aminoacid residue 128 of SEQ ID NO: 1. Even more preferably, the isolatedpolypeptide comprises a codon mutation that results in a substitutionmutation A110W, V128F or V128F/A110P of SEQ ID NO: 1.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation differentially responds to non-ecdysteroiddiacylhydrazine ligands. Preferably, the isolated polypeptide comprisesa codon mutation that results in a substitution of an amino acid residueat a position equivalent or analogous to amino acid residues 52, 95,109, 125 or 132 of SEQ ID NO: 1. More preferably, the isolatedpolypeptide comprises a codon mutation that results in a substitution ofa) a proline residue at a position equivalent or analogous to amino acidresidue 52 of SEQ ID NO: 1, b) a histidine or methionine residue residueat a position equivalent or analogous to amino acid residue 95 of SEQ IDNO: 1, c) a leucine residue at a position equivalent or analogous toamino acid residue 109 of SEQ ID NO: 1, d) a leucine, tryptophan,arginine, cysteine or proline residue at a position equivalent oranalogous to amino acid residue 125 of SEQ ID NO: 1 or e) a methionineresidue at a position equivalent or analogous to amino acid residue 132of SEQ ID NO: 1. Even more preferably, the isolated polypeptidecomprises a codon mutation that results in a substitution mutation T52P,R95H, R95M, F109L, M125L, M125W, M125R, M125C, M125P or L132M of SEQ IDNO: 1.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation differentially responds to non-ecdysteroiddiacylhydrazine ligands. More preferably the isolated polypeptidecomprises a codon mutation that results in a substitution of a) a lysineor arginine residue at a position equivalent or analogous to amino acidresidue 48 of SEQ ID NO: 1, b) a glycine, glutamine, methionine,arginine or tryptophan residue at a position equivalent or analogous toamino acid residue 52 of SEQ ID NO: 1, c) an isoleucine, glycine,asparagines, serine or valine residue at a position equivalent oranalogous to amino acid residue 125 of SEQ ID NO: 1, d) a glutamic acidresidue at a position equivalent or analogous to amino acid residue 132of SEQ ID NO: 1, e) a lysine, tryptophan or tyrosine residue at aposition equivalent or analogous to amino acid residue 219 of SEQ ID NO:1, f) an arginine or tyrosine residue at a position equivalent oranalogous to amino acid residue 223 of SEQ ID NO: 1 or g) leucine ormethionine residue at a position equivalent or analogous to amino acidresidue 238 of SEQ ID NO: 1. Even more preferably the isolatedpolypeptide comprises a codon mutation that results in a substitutionmutation F48K, F48R, T52G, T52Q, T52M, T52R, T52W, M125I, M125G, M125N,M125S, M125V, L132E, M219K, M219W, M219Y, L223R, L223Y, W238L or W238Mof SEQ ID NO: 1.

The present invention also relates to compositions comprising anisolated polypeptide according to the invention.

Method of Modulating Gene Expression of the Invention

Applicants' invention also relates to methods of modulating geneexpression in a host cell using a gene expression modulation systemaccording to the invention. Specifically, Applicants' invention providesa method of modulating the expression of a gene in a host cellcomprising the steps of: a) introducing into the host cell a geneexpression modulation system according to the invention; and b)introducing into the host cell a ligand; wherein the gene to bemodulated is a component of a gene expression cassette comprising: i) aresponse element comprising a domain recognized by the DNA bindingdomain of the gene expression system; ii) a promoter that is activatedby the transactivation domain of the gene expression system; and iii) agene whose expression is to be modulated, whereby upon introduction ofthe ligand into the host cell, expression of the gene is modulated.

The invention also provides a method of modulating the expression of agene in a host cell comprising the steps of: a) introducing into thehost cell a gene expression modulation system according to theinvention; b) introducing into the host cell a gene expression cassetteaccording to the invention, wherein the gene expression cassettecomprises i) a response element comprising a domain recognized by theDNA binding domain from the gene expression system; ii) a promoter thatis activated by the transactivation domain of the gene expressionsystem; and iii) a gene whose expression is to be modulated; and c)introducing into the host cell a ligand; whereby upon introduction ofthe ligand into the host cell, expression of the gene is modulated.

Applicants' invention also provides a method of modulating theexpression of a gene in a host cell comprising a gene expressioncassette comprising a response element comprising a domain to which theDNA binding domain from the first hybrid polypeptide of the geneexpression modulation system binds; a promoter that is activated by thetransactivation domain of the second hybrid polypeptide of the geneexpression modulation system; and a gene whose expression is to bemodulated; wherein the method comprises the steps of: a) introducinginto the host cell a gene expression modulation system according to theinvention; and b) introducing into the host cell a ligand; whereby uponintroduction of the ligand into the host, expression of the gene ismodulated.

Genes of interest for expression in a host cell using Applicants'methods may be endogenous genes or heterologous genes. Nucleic acid oramino acid sequence information for a desired gene or protein can belocated in one of many public access databases, for example, GENBANK,EMBL, Swiss-Prot, and PIR, or in many biology related journalpublications. Thus, those skilled in the art have access to nucleic acidsequence information for virtually all known genes. Such information canthen be used to construct the desired constructs for the insertion ofthe gene of interest within the gene expression cassettes used inApplicants' methods described herein.

Examples of genes of interest for expression in a host cell usingApplicants' methods include, but are not limited to: antigens producedin plants as vaccines, enzymes like alpha-amylase, phytase, glucanes,xylase and xylanase, genes for resistance against insects, nematodes,fungi, bacteria, viruses, and abiotic stresses, nutraceuticals,pharmaceuticals, vitamins, genes for modifying amino acid content,herbicide resistance, cold, drought, and heat tolerance, industrialproducts, oils, protein, carbohydrates, antioxidants, male sterileplants, flowers, fuels, other output traits, genes encodingtherapeutically desirable polypeptides or products that may be used totreat a condition, a disease, a disorder, a dysfunction, a geneticdefect, such as monoclonal antibodies, enzymes, proteases, cytokines,interferons, insulin, erythropoietin, clotting factors, other bloodfactors or components, viral vectors for gene therapy, virus forvaccines, targets for drug discovery, functional genomics, andproteomics analyses and applications, and the like.

Acceptable ligands are any that modulate expression of the gene whenbinding of the DNA binding domain of the gene expression systemaccording to the invention to the response element in the presence ofthe ligand results in activation or suppression of expression of thegenes. Preferred ligands include an ecdysteroid, such as ecdysone,20-hydroxyecdysone, ponasterone A, muristerone A, and the like,9-cis-retinoic acid, synthetic analogs of retinoic acid,N,N′-diacylhydrazines such as those disclosed in U.S. Pat. Nos.6,013,836; 5,117,057; 5,530,028; 5,378,726; and U.S. patent applicationSer. Nos. 10/775,883 and 10/787,906; dibenzoylalkyl cyanohydrazines suchas those disclosed in European Application No. 461,809;N-alkyl-N,N′-diaroylhydrazines such as those disclosed in U.S. Pat. No.5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those disclosed inEuropean Application No. 234,994; N-aroyl-N-alkyl-N′-aroylhydrazinessuch as those described in U.S. Pat. No. 4,985,461; tetrahydroquinolinessuch as those disclosed in International Application No.PCT/JUS03/00915; each of which is incorporated herein by reference andother similar materials including3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide,oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol,25-epoxycholesterol, T0901317,5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS),7-ketocholesterol-3-sulfate, farnesol, bile acids, 1,1-biphosphonateesters, Juvenile hormone III, and the like.

In a preferred embodiment, the ligand for use in Applicants' method ofmodulating expression of gene is a compound of the formula:

wherein:

-   -   E is a branched (C₄-C₁₂)alkyl or branched (C₄-C₁₂)alkenyl        containing a tertiary carbon or a cyano(C₃-C₁₂)alkyl containing        a tertiary carbon;    -   R¹ is H, Me, Et, i-Pr, F, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl,        CH₂OH, CH₂OMe, CH₂CN, CN, C^(o)CH, 1-propynyl, 2-propynyl,        vinyl, OH, OMe, OEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido,        SCN, or SCHF₂;    -   R² is H, Me, Et, n-Pr, i-Pr, formyl, CF₃, CHF₂, CHCl₂, CH₂F,        CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C^(o)CH, 1-propynyl,        2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe₂,        NEt₂, SMe, SEt, SOCF₃, OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃,        CH═CHCN, allyl, azido, OCF₃, OCHF₂, O-i-Pr, SCN, SCHF₂, SOMe,        NH—CN, or joined with R³ and the phenyl carbons to which R² and        R³ are attached to form an ethylenedioxy, a dihydrofuryl ring        with the oxygen adjacent to a phenyl carbon, or a dihydropyryl        ring with the oxygen adjacent to a phenyl carbon;    -   R³ is H, Et, or joined with R² and the phenyl carbons to which        R² and R³ are attached to form an ethylenedioxy, a dihydrofuryl        ring with the oxygen adjacent to a phenyl carbon, or a        dihydropyryl ring with the oxygen adjacent to a phenyl carbon;    -   R⁴, R⁵, and R⁶ are independently H, Me, Et, F, Cl, Br, formyl,        CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CN, C^(o)CH, 1-propynyl,        2-propynyl, vinyl, OMe, OEt, SMe, or Set.

In another preferred embodiment, the ligand for use in Applicants'method of modulating expression of gene is a compound of the formula:

wherein: R1 R2 R3 R4 1 —CH₂CH₃ —OCH₃ —CH₃ —CH₃ 2 —CH₃ —CH₂CH₃ —CH3 —CH₃3 —CH₃ -i-Pr —CH3 —CH₃

In another preferred embodiment, the ligand for use in Applicants'method of modulating expression of gene is a compound of the formula:

wherein: R1 R2 R3 R4 R5 R6 E 1 —CH₂CH₃ —OCH₂CH₂O— —OCH₃ —CH₃ —OCH₃—CH(CH₂CH₃)tBu 2 H H —CH₂CH₃ —CH₃ H —CH₃ —CH(nBu)tBu 3 —CH₂CH₃—OCH₂CH₂O— —OCH₃ —CH₃ —OCH₃ —CH(tBu)CH═C(CH₃)tBu

In a further preferred embodiment, the ligand for use in Applicants'method of modulating expression of gene is a compound of the formula:

wherein: R1 R2 R3 1 F F 3-F-4-CH₃-Ph- 2 F F 3-CH₃-4-F-Ph-

In another preferred embodiment, the ligand for use in Applicants'method of modulating expression of gene is an ecdysone,20-hydroxyecdysone, ponasterone A, muristerone A, an oxysterol, a 22(R)hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol,T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS),7-ketocholesterol-3-sulfate, farnesol, bile acids, 1,1-biphosphonateesters, or Juvenile hormone III.

In another preferred embodiment, a second ligand may be used in additionto the first ligand discussed above in Applicants' method of modulatingexpression of a gene. Preferably, this second ligand is 9-cis-retinoicacid or a synthetic analog of retinoic acid.

Host Cells and Non-Human Organisms of the Invention

As described above, the gene expression modulation system of the presentinvention may be used to modulate gene expression in a host cell.Expression in transgenic host cells may be useful for the expression ofvarious genes of interest. Applicants' invention provides for modulationof gene expression in prokaryotic and eukaryotic host cells. Expressionin transgenic host cells is useful for the expression of variouspolypeptides of interest including but not limited to antigens producedin plants as vaccines, enzymes like alpha-amylase, phytase, glucanes,xylase and xylanase, genes for resistance against insects, nematodes,fungi, bacteria, viruses, and abiotic stresses, antigens,nutraceuticals, pharmaceuticals, vitamins, genes for modifying aminoacid content, herbicide resistance, cold, drought, and heat tolerance,industrial products, oils, protein, carbohydrates, antioxidants, malesterile plants, flowers, fuels, other output traits, therapeuticpolypeptides, pathway intermediates; for the modulation of pathwaysalready existing in the host for the synthesis of new productsheretofore not possible using the host; cell based assays; functionalgenomics assays, biotherapeutic protein production, proteomics assays,and the like. Additionally the gene products may be useful forconferring higher growth yields of the host or for enabling analternative growth mode to be utilized.

Thus, Applicants' invention provides an isolated host cell comprising agene expression system according to the invention. The present inventionalso provides an isolated host cell comprising a gene expressioncassette according to the invention. Applicants' invention also providesan isolated host cell comprising a polynucleotide or a polypeptideaccording to the invention. The present invention also relates to a hostcell transfected with an expression vector according to the invention.The host cell may be a bacterial cell, a fungal cell, a nematode cell,an insect cell, a fish cell, a plant cell, an avian cell, an animalcell, or a mammalian cell. In still another embodiment, the inventionrelates to a method for producing a nuclear receptor ligand bindingdomain comprising a substitution mutation, wherein the method comprisesculturing the host cell as described above in culture medium underconditions permitting expression of a polynucleotide encoding thenuclear receptor ligand binding domain comprising a substitutionmutation, and isolating the nuclear receptor ligand binding domaincomprising a substitution mutation from the culture.

In a specific embodiment, the isolated host cell is a prokaryotic hostcell or a eukaryotic host cell. In another specific embodiment, theisolated host cell is an invertebrate host cell or a vertebrate hostcell. Preferably, the host cell is selected from the group consisting ofa bacterial cell, a fungal cell, a yeast cell, a nematode cell, aninsect cell, a fish cell, a plant cell, an avian cell, an animal cell,and a mammalian cell. More preferably, the host cell is a yeast cell, anematode cell, an insect cell, a plant cell, a zebrafish cell, a chickencell, a hamster cell, a mouse cell, a rat cell, a rabbit cell, a catcell, a dog cell, a bovine cell, a goat cell, a cow cell, a pig cell, ahorse cell, a sheep cell, a simian cell, a monkey cell, a chimpanzeecell, or a human cell. Examples of preferred host cells include, but arenot limited to, fungal or yeast species such as Aspergillus,Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterialspecies such as those in the genera Synechocystis, Synechococcus,Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces,Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes,Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella;plant species selected from the group consisting of an apple,Arabidopsis, bajra, banana, barley, beans, beet, blackgram, chickpea,chili, cucumber, eggplant, favabean, maize, melon, millet, mungbean,oat, okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple,Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash, sugarcane,sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon,and wheat; animal; and mammalian host cells.

In a specific embodiment, the host cell is a yeast cell selected fromthe group consisting of a Saccharomyces, a Pichia, and a Candida hostcell.

In another specific embodiment, the host cell is a Caenorhabdus elegansnematode cell.

In another specific embodiment, the host cell is an insect cell.

In another specific embodiment, the host cell is a plant cell selectedfrom the group consisting of an apple, Arabidopsis, bajra, banana,barley, beans, beet, blackgram, chickpea, chili, cucumber, eggplant,favabean, maize, melon, millet, mungbean, oat, okra, Panicum, papaya,peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin,rice, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweetpotato, tea, tomato, tobacco, watermelon, and wheat cell.

In another specific embodiment, the host cell is a zebrafish cell.

In another specific embodiment, the host cell is a chicken cell.

In another specific embodiment, the host cell is a mammalian cellselected from the group consisting of a hamster cell, a mouse cell, arat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goatcell, a cow cell, a pig cell, a horse cell, a sheep cell, a monkey cell,a chimpanzee cell, and a human cell.

Host cell transformation is well known in the art and may be achieved bya variety of methods including but not limited to electroporation, viralinfection, plasmid/vector transfection, non-viral vector mediatedtransfection, Agrobacterium-mediated transformation, particlebombardment, and the like. Expression of desired gene products involvesculturing the transformed host cells under suitable conditions andinducing expression of the transformed gene. Culture conditions and geneexpression protocols in prokaryotic and eukaryotic cells are well knownin the art (see General Methods section of Examples). Cells may beharvested and the gene products isolated according to protocols specificfor the gene product.

In addition, a host cell may be chosen which modulates the expression ofthe inserted polynucleotide, or modifies and processes the polypeptideproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification [e.g., glycosylation,cleavage (e.g., of signal sequence)] of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce a non-glycosylated coreprotein product. However, a polypeptide expressed in bacteria may not beproperly folded. Expression in yeast can produce a glycosylated product.Expression in eukaryotic cells can increase the likelihood of “native”glycosylation and folding of a heterologous protein. Moreover,expression in mammalian cells can provide a tool for reconstituting, orconstituting, the polypeptide's activity. Furthermore, differentvector/host expression systems may affect processing reactions, such asproteolytic cleavages, to a different extent.

Applicants' invention also relates to a non-human organism comprising anisolated host cell according to the invention. In a specific embodiment,the non-human organism is a prokaryotic organism or a eukaryoticorganism. In another specific embodiment, the non-human organism is aninvertebrate organism or a vertebrate organism.

Preferably, the non-human organism is selected from the group consistingof a bacterium, a fungus, a yeast, a nematode, an insect, a fish, aplant, a bird, an animal, and a mammal. More preferably, the non-humanorganism is a yeast, a nematode, an insect, a plant, a zebrafish, achicken, a hamster, a mouse, a rat, a rabbit, a cat, a dog, a bovine, agoat, a cow, a pig, a horse, a sheep, a simian, a monkey, or achimpanzee.

In a specific embodiment, the non-human organism is a yeast selectedfrom the group consisting of Saccharomyces, Pichia, and Candida.

In another specific embodiment, the non-human organism is a Caenorhabduselegans nematode.

In another specific embodiment, the non-human organism is a plantselected from the group consisting of an apple, Arabidopsis, bajra,banana, barley, beans, beet, blackgram, chickpea, chili, cucumber,eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum,papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato,pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet,sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat.

In another specific embodiment, the non-human organism is a Mus musculusmouse.

Measuring Gene Expression/Transcription

One useful measurement of Applicants' methods of the invention is thatof the transcriptional state of the cell including the identities andabundances of RNA, preferably mRNA species. Such measurements areconveniently conducted by measuring cDNA abundances by any of severalexisting gene expression technologies.

Nucleic acid array technology is a useful technique for determiningdifferential mRNA expression. Such technology includes, for example,oligonucleotide chips and DNA microarrays. These techniques rely on DNAfragments or oligonucleotides which correspond to different genes orcDNAs which are immobilized on a solid support and hybridized to probesprepared from total mRNA pools extracted from cells, tissues, or wholeorganisms and converted to cDNA. Oligonucleotide chips are arrays ofoligonucleotides synthesized on a substrate using photolithographictechniques. Chips have been produced which can analyze for up to 1700genes. DNA microarrays are arrays of DNA samples, typically PCR productsthat are robotically printed onto a microscope slide. Each gene isanalyzed by a full or partial-length target DNA sequence. Microarrayswith up to 10,000 genes are now routinely prepared commercially. Theprimary difference between these two techniques is that oligonucleotidechips typically utilize 25-mer oligonucleotides which allowfractionation of short DNA molecules whereas the larger DNA targets ofmicroarrays, approximately 1000 base pairs, may provide more sensitivityin fractionating complex DNA mixtures.

Another useful measurement of Applicants' methods of the invention isthat of determining the translation state of the cell by measuring theabundances of the constituent protein species present in the cell usingprocesses well known in the art.

Where identification of genes associated with various physiologicalfunctions is desired, an assay may be employed in which changes in suchfunctions as cell growth, apoptosis, senescence, differentiation,adhesion, binding to a specific molecules, binding to another cell,cellular organization, organogenesis, intracellular transport, transportfacilitation, energy conversion, metabolism, myogenesis, neurogenesis,and/or hematopoiesis is measured.

In addition, selectable marker or reporter gene expression may be usedto measure gene expression modulation using Applicants' invention.

Other methods to detect the products of gene expression are well knownin the art and include Southern blots (DNA detection), dot or slot blots(DNA, RNA), northern blots (RNA), RT-PCR(RNA), western blots(polypeptide detection), and ELISA (polypeptide) analyses. Although lesspreferred, labeled proteins can be used to detect a particular nucleicacid sequence to which it hybridizes.

In some cases it is necessary to amplify the amount of a nucleic acidsequence. This may be carried out using one or more of a number ofsuitable methods including, for example, polymerase chain reaction(“PCR”), ligase chain reaction (“LCR″), strand displacementamplification (“SDA”), transcription-based amplification, and the like.PCR is carried out in accordance with known techniques in which, forexample, a nucleic acid sample is treated in the presence of a heatstable DNA polymerase, under hybridizing conditions, with one pair ofoligonucleotide primers, with one primer hybridizing to one strand(template) of the specific sequence to be detected. The primers aresufficiently complementary to each template strand of the specificsequence to hybridize therewith. An extension product of each primer issynthesized and is complementary to the nucleic acid template strand towhich it hybridized. The extension product synthesized from each primercan also serve as a template for further synthesis of extension productsusing the same primers. Following a sufficient number of rounds ofsynthesis of extension products, the sample may be analyzed as describedabove to assess whether the sequence or sequences to be detected arepresent.

Ligand Screening Assays

The present invention also relates to methods of screening for acompound that induces or represses transactivation of a nuclear receptorligand binding domain comprising a substitution mutation in a cell bycontacting a nuclear receptor ligand binding domain with a candidatemolecule and detecting reporter gene activity in the presence of theligand. Candidate compounds may be either agonists or antagonists of thenuclear receptor ligand binding domain. In a preferred embodiment, thenuclear receptor ligand binding domain is expressed from apolynucleotide in the cell and the transactivation activity (i.e.,expression or repression of a reporter gene) or compound bindingactivity is measured.

Accordingly, in addition to rational design of agonists and antagonistsbased on the structure of a nuclear receptor ligand binding domain, thepresent invention contemplates an alternative method for identifyingspecific ligands of a nuclear receptor ligand binding domain usingvarious screening assays known in the art.

Any screening technique known in the art can be used to screen for GroupH nuclear receptor ligand binding domain agonists or antagonists. Forexample, a suitable cell line comprising a nuclear receptor-based geneexpression system according to the invention can be transfected with agene expression cassette encoding a marker gene operatively linked to aninducible or repressible promoter. The transfected cells are thenexposed to a test solution comprising a candidate agonist or antagonistcompound, and then assayed for marker gene expression or repression. Thepresence of more marker gene expression relative to control cells notexposed to the test solution is an indication of the presence of anagonist compound in the test solution. Conversely, the presence of lessmarker gene expression relative to control cells not exposed to the testsolution is an indication of the presence of an antagonist compound inthe test solution.

The present invention contemplates screens for small molecule ligands orligand analogs and mimics, as well as screens for natural ligands thatbind to and agonize or antagonize a Group H nuclear receptor ligandbinding domain according to the invention in vivo. For example, naturalproducts libraries can be screened using assays of the invention formolecules that agonize or antagonize nuclear receptor-based geneexpression system activity.

Identification and screening of antagonists is further facilitated bydetermining structural features of the protein, e.g., using X-raycrystallography, neutron diffraction, nuclear magnetic resonancespectrometry, and other techniques for structure determination. Thesetechniques provide for the rational design or identification of agonistsand antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” [Scott and Smith, 1990, Science 249:386-390 (1990); Cwirla, et al., Proc. Natl. Acad. Sci., 87: 6378-6382(1990); Devlin et al., Science, 249: 404-406 (1990)], very largelibraries can be constructed (10⁶-10⁸ chemical entities). A secondapproach uses primarily chemical methods, of which the Geysen method[Geysen et al., Molecular Immunology 23: 709-715 (1986); Geysen et al.J. Immunologic Method 102: 259-274 (1987)] and the method of Fodor etal. [Science 251: 767-773 (1991)] are examples. Furka et al. [14thInternational Congress of Biochemistry, Volume 5, Abstract FR:013(1988); Furka, Int. J. Peptide Protein Res. 37:487-493 (1991)], Houghton[U.S. Pat. No. 4,631,211, issued December 1986] and Rutter et al. [U.S.Pat. No. 5,010,175, issued Apr. 23, 1991] describe methods to produce amixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries [Needels et al., Proc. Natl.Acad. Sci. USA 90: 107004 (1993); Ohlmeyer et al., Proc. Natl. Acad.Sci. USA 90: 10922-10926 (1993); Lam et al., International PatentPublication No. WO 92/00252; Kocis et al., International PatentPublication No. WO 9428028, each of which is incorporated herein byreference in its entirety], and the like can be used to screen forcandidate ligands according to the present invention.

The screening can be performed with recombinant cells that express anuclear receptor ligand binding domain according to the invention, oralternatively, using purified protein, e.g., produced recombinantly, asdescribed above. For example, labeled, soluble nuclear receptor ligandbinding domains can be used to screen libraries, as described in theforegoing references.

In one embodiment, a Group H nuclear receptor ligand binding domainaccording to the invention may be directly labeled. In anotherembodiment, a labeled secondary reagent may be used to detect binding ofa nuclear receptor ligand binding domain of the invention to a moleculeof interest, e.g., a molecule attached to a solid phase support. Bindingmay be detected by in situ formation of a chromophore by an enzymelabel. Suitable enzymes include, but are not limited to, alkalinephosphatase and horseradish peroxidase. In a further embodiment, atwo-color assay, using two chromogenic substrates with two enzyme labelson different acceptor molecules of interest, may be used. Cross-reactiveand singly reactive ligands may be identified with a two-color assay.

Other labels for use in the invention include colored latex beads,magnetic beads, fluorescent labels (e.g., fluorescene isothiocyanate(FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelatedlanthanide series salts, especially Eu³⁺, to name a few fluorophores),chemiluminescent molecules, radioisotopes, or magnetic resonance imaginglabels. Two-color assays may be performed with two or more colored latexbeads, or fluorophores that emit at different wavelengths. Labeledmolecules or cells may be detected visually or by mechanical/opticalmeans. Mechanical/optical means include fluorescence activated sorting,i.e., analogous to FACS, and micromanipulator removal means.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention.

EXAMPLES

Applicants have developed a CfEcR homology model and have used thishomology model together with a published Chironomous tetans ecdysonereceptor (“CtEcR”) homology model (Wurtz et al., 2000) to identifycritical residues involved in binding to ecdysteroids andnon-ecdysteroids. The synthetic non-steroid, diacylhydrazines, have beenshown to bind lepidopteran EcRs with high affinity and induce precociousincomplete molt in these insects (Wing et al., 1988) and several ofthese compounds are currently marketed as insecticides. The ligandbinding cavity of EcRs has evolved to fit the long backbone structuresof ecdysteroids such as 20E. The diacylhydrazines have a compactstructure compared to ecdysteroids and occupy only the bottom part ofthe EcR binding pocket. This leaves a few critical residues at the toppart of the binding pocket that make contact with ecdysteroids but notwith non-ecdysteroids such as diacylhydrazines. Applicants madesubstitution mutations of the residues that make contact withecdysteroids and/or non-ecdysteroids and determined the mutationaleffect on ligand binding. Applicants describe herein substitutionmutations at several of these residues and have identified severalclasses of substitution mutant receptors based upon their binding andtransactivation characteristics. Applicants' novel substitution mutatednuclear receptor polynucleotides and polypeptides are useful in anuclear receptor-based inducible gene modulation system for variousapplications including gene therapy, expression of proteins of interestin host cells, production of transgenic organisms, and cell-basedassays.

General Methods

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) andby T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984)and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of host cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma ChemicalCompany (St. Louis, Mo.) unless otherwise specified.

Manipulations of genetic sequences may be accomplished using the suiteof programs available from the Genetics Computer Group Inc. (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).Where the GCG program “Pileup” is used the gap creation default value of12, and the gap extension default value of 4 may be used. Where the CGC“Gap” or “Bestfit” program is used the default gap creation penalty of50 and the default gap extension penalty of 3 may be used. In any casewhere GCG program parameters are not prompted for, in these or any otherGCG program, default values may be used.

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “μg” means microgram(s), “mg” meansmilligram(s), “A” means adenine or adenosine, “T” means thymine orthymidine, “G” means guanine or guanosine, “C” means cytidine orcytosine, “x g” means times gravity, “nt” means nucleotide(s), “aa”means amino acid(s), “bp” means base pair(s), “kb” means kilobase(s),“k” means kilo, “μ” means micro, and “° C.” means degrees Celsius.

Example 1

This Example describes the construction of several gene expressioncassettes comprising novel substitution mutant Group H nuclear receptorpolynucleotides and polypeptides of the invention for use in a nuclearreceptor-based inducible gene expression system. Applicants constructedgene expression cassettes based on the spruce budworm Choristoneurafumiferana EcR (“CfEcR). The prepared receptor constructs comprise aligand binding domain of either an EcR or a chimera of Homo sapiensRXRβ-LmRXR; and a GAL4 DNA binding domain (DBD) or a VP16transactivation domain (AD). The reporter constructs include thereporter gene luciferase operably linked to a synthetic promoterconstruct that comprises a GAL4 response element to which the Gal4 DBDbinds. Various combinations of these receptor and reporter constructswere cotransfected into mammalian cells as described in Examples 2-5infra.

Gene Expression Cassettes: Ecdysone receptor-based gene expressioncassettes (switches) were constructed as followed, using standardcloning methods available in the art. The following is a briefdescription of preparation and composition of each switch used in theExamples described herein.

1.1—GAL4CfEcR-DEF/VP16-βRXREF-LmRXREF: The wild-type D, E, and F domainsfrom spruce budworm Choristoneura fumiferana EcR (“CfEcR-DEF”; SEQ IDNO: 21) were fused to a GAL4 DNA binding domain (“Gal4DNABD” or “Gal4DBD”; SEQ ID NO: 6) and placed under the control of a CMV promoter (SEQID NO: 2). Helices 1 through 8 of the EF domains from Homo sapiens RXRβ(“HsRXRβ-EF”; nucleotides 1465 of SEQ ID NO: 3) and helices 9 through 12of the EF domains of Locusta migratoria Ultraspiracle Protein(“LmRXR-EF”; nucleotides 403-630 of SEQ ID NO: 23) were fused to thetransactivation domain from VP16 (“VP16AD”; SEQ ID NO: 12) and placedunder the control of an SV40e promoter (SEQ ID NO: 22). Five consensusGAL4 response element binding sites (“5XGAL4RE”; comprising 5 copies ofa GAL4RE comprising SEQ ID NO: 19) were fused to a synthetic TATAminimal promoter (SEQ ID NO: 24) and placed upstream of the luciferasereporter gene (SEQ ID NO: 25).

1.2—GAL4/mutantCfEcR-DEFNP16-βRXREF-LmRXREF: This construct was preparedin the same way as in switch 1.1 above except wild-type CfEcR-DEF wasreplaced with mutant CfEcR-DEF comprising a ligand binding domaincomprising a substitution mutation selected from Table 1 below.

TABLE 1 Substitution Mutants of Choristoneura fumiferana EcdysoneReceptor (“CfEcR”) Ligand Binding Domain (LBD). Corresponding aminoCfEcR LBD Resulting “WT to Mutant” Amino acid in full length CfEcRMutation Acid Substitution (SEQ ID NO: 26) F48Y Phenylalanine (F) toTyrosine (Y) 331 F48W Phenylalanine (F) to Tryptophan (W) 331 F48LPhenylalanine (F) to Leucine (L) 331 F48N Phenylalanine (F) toAsparagine (N) 331 F48R Phenylalanine (F) to Arginine (R) 331 F48KPhenylalanine (F) to Lysine (K) 331 I51N Isoleucine (I) to Asparagine(N) 334 I51L Isoleucine (I) to Leucine (L) 334 I51M Isoleucine (I) toMethionine (M) 334 T52M Threonine (T) to Methionine (M) 335 T52RThreonine (T) to Arginine (R) 335 T52W Threonine (T) to Tryptophan (W)335 T52G Threonine (T) to Glycine (G) 335 T52Q Threonine (T) toGlutamine (Q) 335 T52E Threonine (T) to Glutamic Acid (E) 335 T52PThreonine (T) to Proline (P) 335 M54W Methionine (M) to Tryptophan (W)337 M54T Methionine (M) to Threonine (T) 337 M92L Methionine (M) toLeucine (L) 375 M92E Methionine (M) to Glutamic Acid (E) 375 R95HArginine (R) to Histidine (H) 378 R95M Arginine (R) to Methionine (M)378 R95W Arginine (R) to Tryptophan (W) 378 V96L Valine (V) to Leucine(L) 379 V96W Valine (V) to Tryptophan (W) 379 V96S Valine (V) to Serine(S) 379 V96E Valine (V) to Glutamic Acid (E) 379 V107I Valine (V) toIsoleucine (I) 390 F109L Phenlyalanine (F) to Leucine (L) 392 F109PPhenylalanine (F) to Proline (P) 392 F109W Phenylalanine (F) toTryptophan (W) 392 F109M Phenylalanine (F) to Methionine (M) 392 F109NPhenylalanine (F) to Asparagine (N) 392 A110E Alanine (A) to GlutamaticAcid (E) 393 A110N Alanine (A) to Asparagine (N) 393 A110W Alanine (A)to Tryptophan (W) 393 N119F Asparagine (N) to Phenylalanine (F) 402Y120W Tyrosine (Y) to Tryptophan (W) 403 Y120M Tyrosine (Y) toMethionine (M) 403 M125E Methionine (M) to Glutamic Acid (E) 408 M125PMethionine (M) to Proline (P) 408 M125R Methionine (M) to Arginine (R)408 M125L Methionine (M) to Leucine (L) 408 M125C Methionine (M) toCysteine (C) 408 M125W Methionine (M) to Tryptophan (W) 408 M125GMethionine (M) to Glycine (G) 408 M125I Methionine (M) to Isoleucine (I)408 M125N Methionine (M) to Asparagine (N) 408 M125S Methionine (M) toSerine (S) 408 M125V Methionine (M) to Valine (V) 408 V128F Valine (V)to Phenylalanine (F) 411 L132M Leucine (L) to Methionine (M) 415 L132NLeucine (L) to Asparagine (N) 415 L132V Leucine (L) to Valine (V) 415L132E Leucine (L) to Glutamic Acid (E) 415 R175E Arginine (R) toGlutamic Acid (E) 458 M219K Methionine (M) to Lysine (K) 502 M219WMethionine (M) to Tryptophan (W) 502 M219Y Methionine (M) to Tyrosine(Y) 502 M219A Methionine (M) to Alanine (A) 502 L223K Leucine (L) toLysine (K) 506 L223R Leucine (L) to Arginine (R) 506 L223Y Leucine (L)to Tyrosine (Y) 506 L234M Leucine (L) to Methionine (M) 517 L234ILeucine (L) to Isoleucine (I) 517 L234R Leucine (L) to Arginine (R) 517L234W Leucine (L) to Tryptophan (W) 517 W238P Tryptophan (W) to Proline(P) 521 W238E Tryptophan (W) to Glutamic Acid (E) 521 W238Y Tryptophan(W) to Tyrosine (Y) 521 W238L Tryptophan (W) to Leucine (L) 521 W238MTryptophan (W) to Methionine (M) 521 T52V and Threonine (T) to Valine(V) and 335 and 393, respectively A110P double mutant Alanine (A) toProline (P), respectively N119F and Asparagine (N) to Phenylalanine (F)402 and 379, respectively V96T double and Valine (V) to Threonine (T),mutant respectively V128F and Valine (V) to Phenylalanine (F) and 411and 393, respectively A110P double Alanine (A) to Proline (P),respectively mutant T52V, V107I Threonine (T) to Valine (V), 335, 390and 458, and R175E triple Valine (V) to Isoleucine (I) and respectivelymutant Arginine (R) to Glutamic Acid (E), respectively T52A, V107IThreonine (T) to Alanine (A), 335, 390 and 458, and R175E triple Valine(V) to Isoleucine (I) and respectively mutant Arginine (R) to GlutamicAcid (E), respectively V96A, V107I Valine (V) to Alanine (A), 379, 390and 458, and R175E triple Valine (V) to Isoleucine (I) and respectivelymutant Arginine (R) to Glutamic Acid (E), respectively V96T, V107IValine (V) to Threonine (T), 379, 390 and 458, and R175E triple Valine(V) to Isoleucine (I) and respectively mutant Arginine (R) to GlutamicAcid (E), respectively V107I, Y127E Valine (V) to Isoleucine (I),Tyrosine 390, 410 and 393, and A110P triple (Y) to Glutamic Acid (E) andAlanine respectively mutant (A) to Proline (P), respectively V107I,Y127E Valine (V) to Isoleucine (I), Tyrosine 390, 410 and 458, and R175Etriple (Y) to Glutamic Acid (E) and Arginine respectively mutant (R) toGlutamic Acid (E), respectively V107I, A110P Valine (V) to Isoleucine(I), 390, 393, and 458, and R175E triple Alanine (A) to Proline (P) andArginine respectively mutant (R) to Glutamic Acid (E), respectivelyV107I, Y127E, Valine (V) to Isoleucine (I), Tyrosine 390, 410 and 335,T52V triple (Y) to Glutamic Acid (E), and respectively mutant Threonine(T) to Valine (V) V107I/Y127E/G Valine (V) to Isoleucine (I), Tyrosine390, 410, and 542, (Y) to Glutamic Acid (E), and Glycine, respectivelyrespectively

Construction of Ecdysone Receptor Ligand Binding Domains Comprising aSubstitution Mutation:

In an effort to modify EcR ligand binding, residues within the EcRligand binding domains that were predicted to be important for ligandbinding based upon a molecular modeling analysis were mutated in EcRsfrom three different classes of organisms. Table 1 indicates the aminoacid residues within the ligand binding domain of CfEcR (LepidopteranEcR) (SEQ ID NO: 1) that were mutated and examined for modification ofecdysteroid and non-ecdysteroid binding.

Each one of the amino acid substitution mutations listed in Table 1 wasconstructed in an EcR cDNA by PCR mediated site-directed mutagenesis. Inaddition to the many single mutation point mutations made, two differentdouble point mutant CfEcRs were also made: one comprising both the V128Fand A110P substitutions (V128F/A110P), and a second comprising both theN119F and V96T substitutions (N119F/V96T). Three different triple pointmutant CfEcRs were also made: one comprising the V1071, Y127E and A110Psubstitutions (V107I/Y127E/A110P), the second comprising the V1071,Y127E and T52V substitutions (V107I/Y127E/T52V), and the thirdcomprising the V1071 and Y127E substitutions and a glycine (G) insertion(V107I/Y127E/259G) (SEQ ID NO: 1).

PCR site-directed mutagenesis was performed using the Quikchangesite-directed mutagenesis kit (Stratagene, La Jolla, Calif.) using thereaction conditions and cycling parameters as follows. PCR site-directedmutagenesis was performed using 1× reaction buffer (supplied bymanufacturer), 50 ng of dsDNA template, 125 ng of forward primer (FP),125 ng of reverse complementary primer (RCP), and 1 μL of dNTP mix(supplied by manufacturer) in a final reaction volume of 50 μL. Theforward primer and reverse complementary primer used to produce each EcRmutant are presented in Table 2. The cycling parameters used consistedof one cycle of denaturing at 95° C. for 30 seconds, followed by 16cycles of denaturating at 95° C. for 30 seconds, annealing at 55° C. for1 minute, and extending at 68° C. for 22 minutes.

TABLE 2 PCR Primers for Substitution Mutant CfEcR Ligand Binding DomainConstruction PRIMER MUTANT (SEQ ID NO:) PRIMER NUCLEOTTDE SEQUENCE(5′ TO 3′) F48Y FP gtcggacactccctaccgccagatcacag (SEQ ID NO:27) F48Y RCPctgtgatctggcggtagggagtgtccgac (SEQ ID NO:28) F48W FPgtcggacactccctggcgccagatcacagag (SEQ ID NO:29) F48W RCPctctgtgatctggcgccagggagtgtccgac (SEQ ID NO:30) F48L FPgtcggacactcccttgcgccagatcacag (SEQ ID NO:31) F48L RCPctgtgatctggcgcaagggagtgtccgac (SEQ ID NO:32) F48N FPgaggctgacactcccaaccgccagatcacagag (SEQ ID NO:33) F48N RCPctctgtgatctggcggttgggagtgtcagcctc (SEQ ID NO:34) F48R FPgtcggacactccccgccgccagatcacag (SEQ ID NO:35) F48R RCPctgtgatctggcggcggggagtgtccgac (SEQ ID NO:36) F48K FPgtcggacactcccaagcgccagatcacag (SEQ ID NO:37) F48K RCPctgtgatctggcgcttgggagtgtccgac (SEQ ID NO:38) I51N FPctcccttccgccagaacacagagatgactatc (SEQ ID NO:39) I51N RCPgatagtcatctctgtgttctggcggaagggag (SEQ ID NO:40) I51L FPctcccttccgccagctcacagagatgac (SEQ ID NO:41) I51L RCPgtcatctctgtgagctggcggaagggag (SEQ ID NO:42) I51M FPcactcccttccgccagatgacagagatgac (SEQ ID NO:43) I51M RCPgtcatctctgtcatctggcggaagggagtg (SEQ ID NO:44) T52M FPcccttccgccagatcatggagatgactatcctcac (SEQ ID NO:45) T52M RCPgtgaggatagtcatctccatgatctggcggaaggg (SEQ ID NO:46) T52R FPcttccgccagatcagagagatgactatcctcac (SEQ ID NO:47) T52R RCPgtgaggatagtcatctctctgatctggcggaag (SEQ ID NO:48) T52W FPctcccttccgccagatctgggagatgactatcctcac (SEQ ID NO:49) T52W RCPgtgaggatagtcatctcccagatctggcggaagggag (SEQ ID NO:50) T52L FPcccttccgccagatcctagagatgactatcctcac (SEQ ID NO:51) T52L RCPgtgaggatagtcatctctaggatctggcggaaggg (SEQ ID NO:52) T52E FPctcccttccgccagatcgaggagatgactatcctcac (SEQ ID NO:53) T52E RCPgtgaggatagtcatctcctcgatctggcggaagggag (SEQ ID NO:54) T52P FPcttccgccagatcccagagatgactatcctc (SEQ ID NO:55) T52P RCPgaggatagtcatctctgggatctggcggaag (SEQ ID NO:56) T52G FPcttccgccagatcggagagatgactatcctcac (SEQ ID NO:57) T52G RCPgtgaggatagtcatctctccgatctggcggaag (SEQ ID NO:58) T52Q FPcttccgccagatccaagagatgactatcctcac (SEQ ID NO:59) T52Q RCPgtgaggatagtcatctcttggatctggcggaag (SEQ ID NO:60) T52V FPcccttccgccagatcgtagagatgactatcctcac (SEQ ID NO:61) T52V RCPgtgaggatagtcatctctacgatctggcggaaggg (SEQ ID NO:62) M54W FPcgccagatcacagagtggactatcctcacggtc (SEQ ID NO:63) M54W RCPgaccgtgaggatagtccactctgtgatctggcg (SEQ ID NO:64) M54T FPccagatcacagagacgactatcctcacggtc (SEQ ID NO:65) M54T RCPgaccgtgaggatagtcgtctctgtgatctgg (SEQ ID NO:66) M92L FPgctcaagtgaggtactgatgctccgagtcg (SEQ ID NO:67) M92L RCPcgactcggagcatcagtacctcacttgagc (SEQ ID NO:68) M92E FPgctcaagtgaggtagagatgctccgagtcgcg (SEQ ID NO:69) M92E RCPcgcgactcggagcatctctacctcacttgagc (SEQ ID NO:70) R95H FPgaggtaatgatgctccacgtcgcgcgacgatac (SEQ ID NO:71) R95H RCPgtatcgtcgcgcgacgtggagcatcattacctc (SEQ ID NO:72) R95M FPgtgaggtaatgatgctcatggtcgcgcgacgatacgatg (SEQ ID NO:73) R95M RCPcatcgtatcgtcgcgcgaccatgagcatcattacctcac (SEQ ID NO:74) R95W FPgtgaggtaatgatgctctgggtcgcgcgacgatacg (SEQ ID NO:75) R95W RCPcgtatcgtcgcgcgacccagagcatcattacctcac (SEQ ID NO:76) V96L FPgtaatgatgctccgactcgcgcgacgatac (SEQ ID NO:77) V96L RCPgtatcgtcgcgcgagtcggagcatcattac (SEQ ID NO:78) V96W FPgaggtaatgatgctccgatgggcgcgacgatacgatg (SEQ ID NO:79) V96W RCPcatcgtatcgtcgcgcccatcggagcatcattacctc (SEQ ID NO:80) V96S FPggtaatgatgctccgatccgcgcgacgatacg (SEQ ID NO:81) V96S RCPcgtatcgtcgcgcggatcggagcatcattacc (SEQ ID NO:82) V96E FPggtaatgatgctccgagaggcgcgacgatacg (SEQ ID NO:83) V96E RCPcgtatcgtcgcgcctctcggagcatcattacc (SEQ ID NO:84) V96T FPggtaatgatgctccgaaccgcgcgacgatacg (SEQ ID NO:85) V96T RCPcgtatcgtcgcgcggttcggagcatcattacc (SEQ ID NO:86) V107I FPgcggcctcagacagtattctgttcgcgaac (SEQ ID NO:87) V107I RCPgttcgcgaacagaatactgtctgaggccgc (SEQ ID NO:88) F109W FPctcagacagtgttctgtgggcgaacaaccaagcg (SEQ ID NO:89) F109W RCPcgcttggttgttcgcccacagaacactgtctgag (SEQ ID NO:90) F109P FPctcagacagtgttctgcccgcgaacaaccaagc (SEQ ID NO:91) F109P RCPgcttggttgttcgcgggcagaacactgtctgag (SEQ ID NO:92) F109L FPcagacagtgttctgttggcgaacaaccaagcg (SEQ ID NO:93) F109L RCPcgcttggttgttcgccaacagaacactgtctg (SEQ ID NO:94) F109M FPcctcagacagtgttctgatggcgaacaaccaagcg (SEQ ID NO:95) F109M RCPcgcttggttgttcgccatcagaacactgtctgagg (SEQ ID NO:96) F109N FPcctcagacagtgttctgaacgcgaacaaccaagcg (SEQ ID NO:97) F109N RCPcgcttggttgttcgcgttcagaacactgtctgagg (SEQ ID NO:98) A110P FPcagacagtgttctgttcccgaacaaccaagcg (SEQ ID NO:99) A110P RCPcgcttggttgttcgggaacagaacactgtctg (SEQ ID NO:100) A110E FPgacagtgttctgttcgagaacaaccaagcgtacac (SEQ ID NO:101) A110E RCPgtgtacgcttggttgttctcgaacagaacactgtc (SEQ ID NO:102) A110N FPcagacagtgttctgttcaacaacaaccaagcgtacactcgcg (SEQ ID NO:103) A110N RCPcgcgagtgtacgcttggttgttgttgaacagaacactgtctg (SEQ ID NO:104) A110W FPcagacagtgttctgttctggaacaaccaagcgtacactc (SEQ ID NO:105) A110W RCPgagtgtacgcttggttgttccagaacagaacactgtctg (SEQ ID NO:106) N119n RANDOM FPgcgtacactcgcgacnnntaccgcaaggctggcatgg (SEQ ID NO:107) N119n RANDOM RCPccatgccagccttgcggtannngtcgcgagtgtacgc (SEQ ID NO:108) Y120W FPcactcgcgacaactggcgcaaggctggcatg (SEQ ID NO:109) Y120W RCPcatgccagccttgcgccagttgtcgcgagtg (SEQ ID NO:110) Y120M FPcactcgcgacaacatgcgcaaggctggcatggcc (SEQ ID NO:111) Y120M RCPggccatgccagccttgcgcatgttgtcgcgagtg (SEQ ID NO:112) M125P FPcaaggctggcccggcctacgtcatcgag (SEQ ID NO:113) M125P RCPctcgatgacgtaggccgggccagccttg (SEQ ID NO:114) M125R FPcaaggctggcagggcctacgtcatcg (SEQ ID NO:115) M125R RCPcgatgacgtaggccctgccagccttg (SEQ ID NO:116) M125E FPgcaaggctggcgaggcctacgtcatcgag (SEQ ID NO:117) M125E RCPctcgatgacgtaggcctcgccagccttgc (SEQ ID NO:118) M125L FPcaaggctggcctggcctacgtcatcg (SEQ ID NO:119) M125L RCPcgatgacgtaggccaggccagccttg (SEQ ID NO:120) M125C FPccgcaaggctggctgcgcctacgtcatcgagg (SEQ ID NO:121) M125C RCPcctcgatgacgtaggcgcagccagccttgcgg (SEQ ID NO:122) M125G FPccgcaaggctggcggggcctacgtcatcg (SEQ ID NO:123) M125G RCPcgatgacgtaggccccgccagccttgcgg (SEQ ID NO:124) M125I FPccgcaaggctggcatagcctacgtcatcg (SEQ ID NO:125) M125I RCPcgatgacgtaggctatgccagccttgcgg (SEQ ID NO:126) M125V FPgcaaggctggcgtggcctacgtcatcg (SEQ ID NO:127) M125V RCPcgatgacgtaggccacgccagccttgc (SEQ ID NO:128) M125W FPgcaaggctggctgggcctacgtcatcgag (SEQ ID NO:129) M125W RCPctcgatgacgtaggcccagccagccttgc (SEQ ID NO:130) Y127E FPcaaggctggcatggccgaggtcatcgagg (SEQ ID NO:131) Y127E RCPcctcgatgacctcggccatgccagccttg (SEQ ID NO:132) V128F FPggctggcatggcctacnnnatcgaggatctactgcacttc (SEQ ID NO:133) V128F RCPgaagtgcagtagatcctcgatnnngtaggccatgccagcc (SEQ ID NO:134) L132M FPgcctacgtcatcgaggatatgctgcacttctgccgg (SEQ ID NO:135) L132M RCPccggcagaagtgcagcatatcctcgatgacgtaggc (SEQ ID NO:136 L132N FPgcctacgtcatcgaggataacctgcacttctgccgg (SEQ ID NO:137) L132N RCPccggcagaagtgcaggttatcctcgatgacgtaggc (SEQ ID NO:138) L132V FPcgtcatcgaggatgtactgcacttctgccg (SEQ ID NO:139) L132V RCPcggcagaagtgcagtacatcctcgatgacg (SEQ ID NO:140) L132E FPgcctacgtcatcgaggatgaactgcacttctgcc (SEQ ID NO:141) L132E RCPggcagaagtgcagttcatcctcgatgacgtaggc (SEQ ID NO:142) M219K FPgcatgcaaaactccaacaagtgcatctccctcaag (SEQ ID NO:143) M219K RCPcttgagggagatgcacttgttggagttttgcatgc (SEQ ID NO:144) M219W FPgcatgcaaaactccaactggtgcatctccctcaagct (SEQ ID NO:145) M219W RCPagcttgagggagatgcaccagttggagttttgcatgc (SEQ ID NO:146) M219Y FPctcggcatgcaaaactccaactattgcatctccctcaagctcaag (SEQ ID NO:147) M219Y RCPcttgagcttgagggagatgcaatagttggagttttgcatgccgag (SEQ ID NO:148) M219A FPcatgcaaaactccaacgcgtgcatctccctcaag (SEQ ID NO:149) M219A RCPcttgagggagatgcacgcgttggagttttgcatg (SEQ ID NO:150) L223K FPctccaacatgtgcatctccaagaagctcaagaacag (SEQ ID NO:151) L223K RCPctgttcttgagcttcttggagatgcacatgttggag (SEQ ID NO:152) L223R FPctccaacatgtgcatctcccgcaagctcaagaacag (SEQ ID NO:153) L223R RCPctgttcttgagcttgcgggagatgcacatgttggag (SEQ ID NO:154) L223Y FPctccaacatgtgcatctcctacaagctcaagaacag (SEQ ID NO:155) L223Y RCPctgttcttgagcttgtaggagatgcacatgttggag (SEQ ID NO:156) L234M FPgctgccgcctttcatggaggagatctgggatg (SEQ ID NO:157) L234M RCPcatcccagatctcctccatgaaaggcggcagc (SEQ ID NO:158) L234I FPgctgccgcctttcattgaggagatctgggatgtg (SEQ ID NO:159) L234I RCPcacatcccagatctcctcaatgaaaggcggcagc (SEQ ID NO:160) L234R FPctgccgcctttccgagaggagatctgggatg (SEQ ID NO:161) L234R RCPcatcccagatctcctctcggaaaggcggcag (SEQ ID NO:162) L234W FPgctgccgcctttctgggaggagatctgggatgtg (SEQ ID NO:163) L234W RCPcacatcccagatctcctcccagaaaggcggcagc (SEQ ID NO:164) W238P FPctcgaggagatcccggatgtggcaggacatg (SEQ ID NO:165) W238P RCPcatgtcctgccacatccgggatctcctcgag (SEQ ID NO:166) W238E FPcctcgaggagatcgaggatgtggcaggacatg (SEQ ID NO:167) W238E RCPcatgtcctgccacatcctcgatctcctcgagg (SEQ ID NO:168) W238L FPctcgaggagatcttggatgtggcaggacatg (SEQ ID NO:169) W238L RCPcatgtcctgccacatccaagatctcctcgag (SEQ ID NO:170) W238M FPcctcgaggagatcatggatgtggcaggacatgtc (SEQ ID NO:171) W238M RCPgacatgtcctgccacatccatgatctcctcgagg (SEQ ID NO:172) W238Y FPcctcgaggagatctacgatgtggcaggacatgtc (SEQ ID NO:173) W238Y RCPgacatgtcctgccacatcgtagatctcctcgagg (SEQ ID NO:174)

The resulting PCR nucleic acid products encoding the mutant EcR ligandbinding domains were then each fused to a GAL4 DNA binding domain asdescribed in Example 1.2 above. The GAL4/mutant EcR receptor constructswere tested for activity by transfecting them into NIH3T3 cells alongwith VP16/βRXREF-LmRXREF and pFRLuc in the presence of various ligands.

The Gal-4-CfEcR-DEF(VYG) mutant was created by inserting an extraglycine at the C-terminal end of EcR substitution mutant V107I/Y127E[CfECR(VY)] by PCR. Essentially, this was done in two steps:PCR-amplification of CfEcR-DEF(VYG) and substitution of the CfEcR(VY) inthe vector GAL4-CfEcR DEF(VY) pBIND 1-9 with the PCR-amplifiedCfEcR-DEF(VYG). The CfEcR-DEF region (with the extra glycine) wasamplified by using the vector GAL4-CfEcR DEF(VY) pBIND 1-9 as templateand the following PCR primers:

5EcR-wt (SEQ ID NO:175) GGAATTCCCGGGGATCCGGCCTGAGTGCGTAGTACCC 3EcR-gly(SEQ ID NO:176) CTCTCTGCGGCCGCCTATCCGAGATTCGTGGGGGACTCGAGGATAG

The PCR product was isolated and digested with Not I (cuts at the 3′end; included in the 3′ PCR primer) and Xma I (cuts at the 5′ end;present in the 5′PCR primer). This product was ligated to the vectorprepared in the following way: GAL4-CfEcR DEF(VY) pBIND 1-9 was digestedwith Xma I and Not I (the digestion removes the CfEcR-DEF (VY) from thevector). The fragments were separated on 1% agarose gel and the slowermigrating vector DNA was purified. After ligation between the vector andthe CfEcR-DEF(VYG) fragment described above, the ligation reaction wastransformed into bacteria. The positive colonies were selected by colonyPCR using the primers mentioned above. The VYG mutations in the selectedclone were confirmed by sequencing.

Example 2

This Example describes the identification of ecdysteroid responsiveCfEcR ligand binding domain substitution mutants that exhibit increasedactivity in response to ecdysteroidal ligand. In an effort to identifysubstitution mutations in the CfEcR that increase ecdysteroidal ligandactivity, Applicants mutated amino acid residues predicted to becritical for ecdysteroid binding and created GAL4/mutantCfEcR-DEF cDNAgene expression cassettes as described in Example 1 above usingPCR-mediated site-directed mutagenesis kit. The mutated and the WT cDNAscorresponding to the various switch constructs outlined above in Examplewere made and tested in GAL4-driven luciferase reporter assays asdescribed below.

Transfections: DNAs were transfected into mouse NIH3T3 cells (ATCC) asfollows. Standard methods for culture and maintenance of the cells werefollowed. Cells were harvested and plated 96-well plates at 2,500 cellsper well, in 50 μL of growth medium containing 10% fetal bovine serum(FBS). Twenty-four hours later, the cells were treated with 35 μL ofserum-free growth medium containing either dimethylsulfoxide (DMSO;control) or a DMSO solution of ligand. The cells were then transfectedusing Superfect™ (Qiagen Inc.) transfection reagent. For each well,0.625 μL of Superfect™ was mixed with 14.2 μL of serum-free growthmedium. 0.16 μg of reporter construct and 0.04 μg of each receptorconstruct were added to the transfection reagent mix. The contents ofthe transfection mix were mixed in a vortex mixer and let stand at roomtemperature for 30 minutes. At the end of incubation, 15 μL oftransfection mix was added to the cells. The cells were maintained at37° C. and 5% CO₂ for 48 hours in 5% FBS.

Ligands: The ecdysteroidal ligands ponasterone A and 20-hydroxyecdysonewere purchased from Sigma Chemical Company and Invitrogen. Thenon-ecdysteroidal diacylhydrazine ligandN-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine(RG-102240, GS®-E ligand) is a synthetic stable ecdyecdysteroid ligandthat was synthesized at Rohm and Haas Company. The non-ecdysteroidal,diacylhydrazine ligands RG-101691, RG-102362, RG-115840, RG-115853,RG-115855, RG-115859 and RG-115898 were synthesized by RheoGene Inc. Thesynthesis of RG-101691, RG-102362, RG-115840, RG-115859 and RG-115898 isdescribed below. The synthesis of RG-115853 and RG-115855 is describedin co-pending U.S. patent application Ser. No. 10/775,883. Thenon-ecdysteroidal tetrahydroquinoline ligands RG-120499 and RG-120500were synthesized by RheoGene, Inc. and were described in co-pending U.S.patent application Ser. No. 10/460,820. All ligands were dissolved inDMSO.

Ligand Synthesis: Preparation of 3,5-Dimethyl-benzoic acidN-tert-butyl-N′-(3-ethyl-2-methyl-benzoyl)-hydrazide (RG-101691)

3-Amino-2-methylbenzoic acid (6.16 g) was heated at reflux for 30minutes in concentrated HBr. The mixture was cooled to 0° C. and treatedwith a solution of NaNO₂ at 0° C. (2.8 g in 5.6 mL H₂O). The resultantdiazonium salt solution was slowly added to a preheated (60-70° C.)solution of CuBr (3.8 g) in 3.2 mL concentrated HBr. After the addition,the mixture was stirred overnight at room temperature and filtered. Therecovered filter cake was washed first with water and then with 10% HCl,and dried in air to yield 6.93 g of 3-bromo-2-methylbenzoic acid as alight purple powdery solid. This material was dissolved in ethylacetate, washed twice with 5% HCl, dried over Na₂SO₄, and recrystallizedfrom 4:1 hexanes:ethyl acetate first at room temperature and then underrefrigeration. ¹H NMR (DMSO, 200 MHz), δ (ppm): 7.72 (dd, 2H), 7.2 (t,1H), 2.5 (s, 3H).

3-Bromo-2-methylbenzoic acid (7.03 g, 32.7 mmol) was refluxed in 10 mLof SOCl₂ (98 mmol) and a drop of DMF for 3 hours. Excess SOCl₂ wasremoved in vacuo. The residue was dissolved in 20 mL of CH₂Cl₂ and addedto an ice-chilled solution of 2-amino-2-methyl-propan-1-ol (8.74 g, 9.36mL) in 20 mL of CH₂Cl₂. The mixture was stirred at room temperature for18 hours and the solvent was removed in vacuo to leave an oily residue.SOCl₂ (7.4 mL, 100 mL, 3 eq.) was added to this residue over a period ofone hour, the mixture was stirred an additional 30 min, and then pouredinto 150 mL of ether. An oily immiscible phase formed and the ether wasdiscarded. The oil was mixed with 100 mL of 20% NaOH, and extracted with3×150 mL portions of ether. The ether extracts were combined, dried overMgSO₄, and the solvent was removed in vacuo to yield a yellow oil.Chromatography on silica gel using 4:1 hexane:ether as eluant yielded4.87 g of 2-(3-bromo-2-methyl-phenyl)-4,4-dimethyl-4,5-dihydro-oxazoleas a colorless oil. (Rf=0.25 (4:1 hexane:ether). ¹H NMR (CDCl₃, 200MHz), δ (ppm): 7.62 (m, 2H), 7.1 (t, 1H), 4.1 (s, 2H), 2.6 (s, 3H), 1.4(s, 6H).

2-(3-bromo-2-methyl-phenyl)-4,4-dimethyl-4,5-dihydro-oxazole (3.4 g,12.7 mmol) was dissolved in 30 mL of ethyl ether under nitrogenatmosphere in a 100 mL round bottom flask equipped with magneticstirring, thermometer, and reflux condenser. Ni(dppp)Cl₂ (100 mg) wasadded and the mixture was cooled to 0° C. in an ice bath. Ethylmagnesium bromide (5.5 mL, 3M in ether) was added, the reaction mixturewas stirred at 0° C. for 30 minutes, at room temperature for 2½ hours,and finally at reflux for 2 hours. The mixture was then cooled to 0° C.,quenched with saturated aqueous NH₄Cl. The organic layer was removed andthe aqueous layer was extracted with ether. The organic phases werecombined and dried over MgSO₄. The solvent was removed in vacuo to give2.84 g 2-(3-ethyl-2-methyl-phenyl)-4,4-dimethyl-4,5-dihydro-oxazole, ¹HNMR (CDCl₃, 200 MHz), δ (ppm): 7.5 (d, 2H), 7.2 (m, 2H), 4.1 (s, 2H),2.7 (m, 2H), 2.45 (s, 3H), 1.4 (s, 6H), 1.2 (t, 3H), Rf=0.25 (4:1hexane:ether), containing ca. 5% original aryl bromide. The oxazolinewas suspended in 100 mL of 6N HCl and refluxed for 5 hours with vigorousstirring. The mixture was allowed to cool to room temperature, whereupon3-ethyl-2-methyl-benzoic acid crystallized: 1.74 g, m.p. 96-98° C., ¹HNMR (CDCl₃, 200 MHz), δ (ppm): 7.85 (d, 1H), 7.4 (d, 1H), 7.22 (t, 1H),2.7 (q, 2H), 2.6 (s, 3H), 1.21 (t, 3H). An additional 110 mg wasrecovered by ether extraction of the aqueous phase.

3-Ethyl-2-methyl-benzoic acid (0.517 g) was refluxed in 3 mL of thionylchloride with a drop of DMF for several hours. Thionyl chloride wasremoved in vacuo to yield 0.89 g (4.48 mmol) of 3-ethyl-2-methyl-benzoylchloride. The acid chloride was dissolved in 5 mL of CH₂Cl₂ and addedslowly and simultaneously but separately with 5 mL of aqueous NaOH (0.30g, 7.5 mmol) to a solution of 3,5-dimethyl-benzoic acidN-tert-butyl-hydrazide (0.96 g, 4.36 mmol) dissolved in 10 mL of CH₂Cl₂prechilled to −5° C. During the addition, the temperature was kept below5° C. The mixture was allowed to warm slowly to room temperature and wasstirred overnight. The organic layer was removed and the aqueous layerwas extracted with CH₂Cl₂. The organic extracts were combined, dried,and solvent was removed in vacuo to give 1.5 g crude product. Thisresidue was extracted with 100 mL of hexanes under reflux, and the hotextract was decanted from an oily residue and allowed to cool to roomtemperature, whereupon 3,5-dimethyl-benzoic acidN-tert-butyl-N′-(3-ethyl-2-methyl-benzoyl)-hydrazide crystallized (0.56g, m.p. 167-169° C., ¹H NMR (CDCl₃, 200 MHz), δ (ppm): 7.43 (s, 1H),7.18 (m, 1H), 7.1 (s, 2H), 7.03 (s, 1H), 7.0 (m, 1H), 6.35 (d, 1H), 2.58(q, 2H), 2.3 (s, 6H), 1.95 (s, 3H), 1.6 (s, 9H), 1.15 (t, 3H).Dissolution of the oily residue and crystallization yielded a secondcrop of less pure material, 0.21 g.

Preparation of 3,5-Dimethyl-benzoic acidN-tert-butyl-N′-(3-isopropyl-2-methyl-benzoyl)-hydrazide (RG102362)

A dry 3-neck 250 mL round bottom flask equipped with magnetic stirringand held under a nitrogen atmosphere was charged with 5.0 g2-(3-bromo-2-methyl-phenyl)-4,4-dimethyl-4,5-dihydro-oxazole, 60 mLanhydrous THF, and 100 mg Ni(dppp)Cl₂. The mixture was cooled to 15° C.,and isopropyl magnesium chloride (11 mL, 2M in ethyl ether) was added. Amild exotherm took place, and the mixture darkened slightly. Thereaction was stirred overnight at room temperature, at which point ¹HNMR indicated 50% completion. Addition of ca 75 mg Ni(dppp)Cl₂ andreflux for 3 hours resulted in no further progression of the reaction.The mixture was cooled to 15° C., and an additional 13 mL of isopropylmagnesium chloride (2M in ethyl ether) and 100 mg of nickel catalystwere added and the mixture was stirred overnight at room temperature.The reaction was quenched with saturated aqueous NH₄Cl, the organiclayer was removed, the aqueous layer was extracted, and the organicphases were combined and dried. The solvent was removed in vacuo toyield 3.84 g crude product as a yellow oil. Column chromatography onsilica gel using 4:1 hexanes:ether as eluant yielded 0.79 g of2-(3-isopropyl-2-methyl-phenyl)-4,4-dimethyl-4,5-dihydro-oxazole as acolorless oil. ¹H NMR (CDCl₃, 200 MHz), δ (ppm): 7.5 (d, 1H), 7.37 (d,1H), 7.22 (t, 1H), 4.13 (s, 2H), 3.23 (m, 1H), 2.5 (s, 3H), 1.45 (s,6H), 1.22 (d, 6H). The oxazoline was suspended in 34 mL of 6N HCl andrefluxed in an oil bath for 6 hours. The mixture was cooled andextracted with CH₂Cl₂. The extract was dried over Na₂SO₄ and evaporatedto yield 0.76 g of 3-isopropyl-2-methyl-benzoic acid, suitably pure forthe next step. ¹H NMR (CDCl₃, 300 MHz), δ (ppm): 7.8 (d, 1H), 7.48 (d,1H), 7.3 (t, 1H), 3.3 (m, 1H), 2.55 (s, 3H), 1.2 (d, 6H).

3-Isopropyl-2-methyl-benzoic acid (0.75 g) was refluxed in ca. 3 mL ofthionyl chloride with a drop of DMF fro several hours and thionylchloride was removed in vacuo to yield 3-isopropyl-2-methylbenzoylchloride. The acid chloride was dissolved in 5 mL of CH₂Cl₂ and addedslowly and simultaneously but separately with 5 mL of aqueous NaOH(0.265 g, 6.6 mmol) to a solution of 3,5-dimethyl-benzoic acidN-tert-butyl-hydrazide (0.973 g, 4.4 mmol) dissolved in 10 mL of CH₂Cl₂prechilled to −5° C. During the addition, the temperature was kept below5° C. The mixture was allowed to warm slowly to room temperature and wasstirred overnight. The organic layer was removed and the aqueous layerwas extracted with CH₂Cl₂. The organic extracts were combined, dried,and solvent was removed in vacuo to give 1.61 g of crude product as ayellow oil. This material was chromatographed on silica gel using 4:1hexanes:ethyl acetate as eluant, and subsequently triturated from 1:1hexane:ether, yielding 3,5-dimethyl-benzoic acidN-tert-butyl-N′-(3-isopropyl-2-methyl-benzoyl)-hydrazide, after arduousremoval of ether in a vacuum oven at 60° C. (0.35 g, m.p. 182.5° C. ¹HNMR (CDCl₃, 200 MHz), (ppm): 7.6 (s, 1H), 7.25 (d, 1H), 7.1 (s, 2H),7.05 (s, 1H), 7.0 (m, 1H), 6.3 (d, 1H), 3.1 (m, 1H), 2.3 (s, 6H), 1.95(s, 3H), 1.6 (s, 9H), 1.18 (m, 6H).

Preparation of 3,5-dimethyl-benzoic acidN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-N-(1-ethyl-2,2-dimethyl-propyl)-hydrazide(RSG115858)

2.38 g (18 mmol) of t-butyl carbazate were dissolved in 50 mL of CH₂Cl₂in a 250 mL round bottom flask and cooled to 0° C. An aqueous K₂CO₃solution was prepared (4.15 g K₂CO₃/35 mL H₂O) and added to the reactionmixture which was again cooled to 0° C. 3.63 g (16 mmol) of5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl chloride were dissolvedin 40 mL of CH₂Cl₂ and added from a separatory funnel, drop-wise over 15min. The reaction mixture was stirred at room temperature for 3 days.The reaction mixture was transferred to a separatory funnel with CH₂Cl₂and H₂O. The water phase was thoroughly extracted with CH₂Cl₂. TheCH₂Cl₂ extract was then extracted with 0.5N HCl, dried, and evaporated.The residue was further dried in a vacuum oven to yield 5.15 g of a tansolid ofN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinecarboxylicacid tert-butyl ester. TLC (1:1 ethyl acetate:hexane) gave a single spotat Rf=0.43 and NMR indicated a very pure product: ¹H NMR (CDCl₃, 500MHz) δ (ppm): 7.5 (br, 1H), 7.0 (br, 1H), 6.75 (d, 2H), 4.28 (br, 4H),2.76 (m, 2H), δ 1.5 (s, 9H), 1.18 (t, 3H).

5.15 g (16 mmol) ofN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinecarboxylicacid tert-butyl ester were added to a 200 mL round bottom flask. About20 mL of trifluoroacetic acid were added and the reaction mixture wasstirred at room temperature for 24 hours. Then about 40 mL of water wereadded, followed by the slow addition of cold 10% NaOH/H₂O, withstirring, until the acid was neutralized (pH ˜14). The reaction mixturewas transferred to a separatory funnel and extracted with ethyl acetateby shaking gently (caution: gas evolution). The ethyl acetate extractwas dried and evaporated to yield 5.51 g of a pale, viscous yellowsemi-solid. The material was then placed in a 50° C. vacuum oven forabout 1 hour to yield 4.62 g of5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid hydrazide. Thet-Boc cleavage is best accomplished with neat trifluoroacetic acid; useof adjunctive solvents always resulted in much lower yields. ¹H NMR(CDCl₃, 500 MHz) δ (ppm): 7.0 (br, 1H), 6.83 (m, 1H), 6.71 (m, 1H), 4.28(br s, 4H), 2.76 (m, 2H), 1.6 (br, 2H), 1.17 (t, 3H).

1.12 g (5.1 mmol) of 5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylicacid hydrazide, 1.37 g (12 mmol) of 2,2 dimethyl pentanone-3, 30 mL ofethanol, and 20 drops of glacial acetic acid were refluxed for 6 hoursto generate 5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid(1-ethyl-2,2-dimethyl-propylidene)-hydrazide, which was used in situ. Tothe cooled reaction mixture, was added 3 mL of glacial acetic acid and0.63 g (10 mmol) of NaCNBH₃. The reaction was stirred at roomtemperature for 24 hours. 25 mL of water were added and most of thealcohol was removed on a rotary evaporator. Then 10% NaOH/H₂O was addeduntil the reaction mixture was basic. The product was extracted withethyl acetate, which was then dried and evaporated to give 1.61 g ofresidue. Pure 5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acidN′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide was obtained (ca. 0.77 g) bycolumn chromatography on silica gel, eluting with 25% ethylacetate/hexane. TLC: Rf=0.53, 1:1 ethyl acetate:hexane). ¹H NMR (CDCl₃,500 MHz) δ (ppm): 7.1 (br s, 1H), 6.8 (d, 1H), 6.7 (d, 1H), 4.27 (m,4H), 2.8 (m, 2H), 2.4 (m, 1H), 1.7 (m, 1H), 1.3 (m, 1H), 1.2 (t, 3H),1.15 (t, 3H), 0.97 (s, 9H).

0.214 (0.70 mmol) of 5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylicacid N′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide, 151 mg (0.9 mmol) of3,5 dimethylbenzoyl chloride, 7 mL of 25% K₂CO₃/H₂O and 7 mL of CH₂Cl₂were added to a 20 mL vial and stirred at room temperature for 24 hours.The reaction mixture was transferred to a separatory funnel, and diluteNaHCO₃ and CH₂Cl₂ were added. The CH₂Cl₂ layer was separated and thewater layer extracted twice with CH₂Cl₂. The CH₂Cl₂ extracts were driedover MgSO₄ and evaporated to yield 0.59 g of a white residue.Purification by column chromatography and elution with 15 mL of 20%ethyl acetate/hexane yielded about 350 mg of 3,5-dimethyl-benzoic acidN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-N-(1-ethyl-2,2-dimethyl-propyl)-hydrazide(95% pure by TLC: Rf=0.56, 1:1 ethyl acetate:hexane). ¹H NMR (CDCl₃, 500MHz) δ (ppm): 7.05 (s, 1H), 7.0 (s, 2H), 6.6 (d, 1H), 6.27 (d, 1H), 4.65(d, 1H), 4.25 (s, 4H), 2.9 (m, 1H), 2.3 (s, 6H), 2.0 (m, 1H), 1.55-1.7(m, 2H), 1.25 (m, 3H), 0.9-1.2 (3s, 9H), 0.9 (t, 3H).

Preparation of 3,5-dimethoxy-4-methyl-benzoic acidN-(1-tert-butyl-3,4,4-trimethyl-pent-2-enyl)-N′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide(RG115898)

2.38 g (18 mmol) of t-butyl carbazate were dissolved in 50 mL of CH₂Cl₂in a 250 mL round bottom flask and cooled to 0° C. An aqueous K₂CO₃solution was prepared (4.15 g K₂CO₃/35 mL H₂O) and added to the reactionmixture which was again cooled to 0° C. 3.63 g (16 mmol) of5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl chloride were dissolvedin 40 mL of CH₂Cl₂ and added from a separatory funnel, drop-wise over 15min. The reaction mixture was stirred at room temperature for 3 days.The reaction mixture was transferred to a separatory funnel with CH₂Cl₂and H₂O. The water phase was thoroughly extracted with CH₂Cl₂. TheCH₂Cl₂ extract was then extracted with 0.5N HCl, dried, and evaporated.The residue was further dried in a vacuum oven to yield 5.15 g of a tansolid ofN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinecarboxylicacid tert-butyl ester. TLC (1:1 ethyl acetate:hexane) gave a single spotat Rf=0.43 and NMR indicated a very pure product: ¹H NMR (CDCl₃, 500MHz) δ (ppm): 7.5 (br, 1H), 7.0 (br, 1H), 6.75 (d, 2H), 4.28 (br, 4H),2.76 (m, 2H), 1.5 (s, 9H), 1.18 (t, 3H).

5.15 g (16 mmol) ofN′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazinecarboxylicacid tert-butyl ester were added to a 200 mL round bottom flask. About20 mL of trifluoroacetic acid were added and the reaction mixture wasstirred at room temperature for 24 hours. Then about 40 mL of water wereadded, followed by the slow addition of cold 10% NaOH/H₂O, withstirring, until the acid was neutralized (pH ˜14). The reaction mixturewas transferred to a separatory funnel and extracted with ethyl acetateby shaking gently (caution: gas evolution). The ethyl acetate extractwas dried and evaporated to yield 5.51 g of a pale, viscous yellowsemi-solid. The material was then placed in a 50° C. vacuum oven forabout 1 hour to yield 4.62 g of5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid hydrazide. Thet-Boc cleavage is best accomplished with neat trifluoroacetic acid; useof adjunctive solvents always resulted in much lower yields. ¹H NMR(CDCl₃, 500 MHz) δ (ppm): 7.0 (br, 1H), 6.83 (m, 1H), 6.71 (m, 1H), 4.28(br s, 4H), 2.76 (m, 2H), 1.6 (br, 2H), 1.17 (t, 3H).

2,2,5,6,6-Pentamethyl-hept-4-en-3-one (1.48 g, 8.1 mmol) was dissolvedin n-butyl alcohol (20 mL). Then5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid hydrazide (1.80g, 8.1 mmol) and 10 drops of glacial acetic acid were added. Thereaction mixture was refluxed for 20 hours (required for completereaction) and monitored by TLC. To a solution of the intermediate5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid(1-tert-butyl-3,4,4-trimethyl-pent-2-enylidene)-hydrazide were added 1.8mL glacial acetic acid and 1.02 g (16.2 mmol) of sodiumcyanoborohydride. The reaction was refluxed for three hours. Thereaction was cooled and 50 mL of water and 10% aqueous NaOH were addeduntil the reaction was basic (pH=ca. 14). Most of the alcohol wasremoved on a rotary evaporator and the residue was extracted with EtOAc.The aqueous extract was dried and concentrated to constant weight,yielding 4 g of a viscous material. 2.3 g pure5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acidN′-(1-tert-butyl-3,4,4-trimethyl-pent-2-enyl)-hydrazide was obtained(yellow oil, R_(f)=0.30 in 25% EtOAc in n-Hexane, yield 73%) by columnchromatography on silica gel. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.42 (br,1H), 6.80 (d, J=8.4 Hz, 1H), 6.71 (d, J=8.4 Hz, 1H), 6.17 (br, 1H), 5.30(dd, J=0.8, 10 Hz, 1H), 4.33-4.29 (m, 4H), 3.68 (d, J=10 Hz, 1H), 2.80(m, 2H), 1.72 (s, 3H), 1.21 (s, 3H), 1.12 (s, 9H), 1.05 (s, 9H).

5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acidN′-(1-tert-butyl-3,4,4-trimethyl-pent-2-enyl)-hydrazide (150 mg, 0.39mmol) and 3,5-dimethoxy-4-methylbenzoyl chloride (83 mg, 0.39 mmol) weredissolved in 5 mL CH₂Cl₂. 5 mL of 25% K₂CO₃ were added, and the reactionmixture was stirred at room temperature overnight. The reaction wasmonitored by TLC. The phases were separated, adding additional CH₂Cl₂and/or water as needed to aid manipulation. The CH₂Cl₂ layer was driedand solvent was removed in vacuo to provide 210 mg of crude product.This material was purified by silica gel column chromatography, elutingwith a step gradient of 10-25% ethyl acetate in hexane to yield3,5-dimethoxy-4-methyl-benzoic acidN-(1-tert-butyl-3,4,4-trimethyl-pent-2-enyl)-N′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazideRG115898 (83 mg, R_(f)=0.19 in 25% ethyl acetate in n-hexane, yield38%). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.19 (s, 1H), 6.75 (d, J=8.0Hz, 1H), 6.69 (s, 2H), 6.61 (d, J=8.0 Hz, 1H), 5.43 (d, J=10.0 Hz, 1H),5.41 (d, 14.4 Hz, 1H), 4.30-4.20 (m, 4H), 3.80 (s, 6H), 2.21-2.15 (m,1H), 2.01 (s, 3H), 1.81 (m, 1H), 1.76-1.64 (m, 1H), 1.06 (s, 9H), 1.00(s, 9H), 0.70 (t, J=7.6 Hz, 3H).

Preparation of 3,5-dimethyl-benzoic acidN-(1-tert-butyl-pentyl)-N′-(4-ethyl-benzoyl)-hydrazide (RG-115840)

2,2-Dimethyl-heptan-3-ol (0.23 mol) was dissolved in 350 mL of CH₂Cl₂ ina 500 mL round bottom flask with a magnetic stirbar. The flask waspartially cooled with ice. 76.6 g (0.355 mol) of pyridiniumchlorochromate was added, while vigorously stirring. The reaction turnedblack and warmed up slightly. The reaction mixture was stirred at roomtemperature for 24 hours. The solution was decanted away from the blacksludge, which was rinsed with hexane. The organic extracts were combinedand chromatographed directly on silica gel. (Note: only silica has beenfound to trap and remove the reduced non-reacted chromium compounds).The product, 2,2-dimethyl-heptan-3-one, eluted with CH₂Cl₂/hexane and ina subsequent 10% ethyl acetate/hexane fraction to yield 29.19 g ofproduct at 88% yield. ¹H NMR (CDCl₃, 500 MHz) δ (ppm): 2.48 (t, 2H),1.54 (m, 2H), 1.28 (m, 2H), 1.13 (s, 9H), 0.90 (m, 3H).

Preparation of 4-ethyl-benzoic acid N′-(1-tert-butyl-pentyl)-hydrazide

4-Ethyl-benzoic acid hydrazide (1.64 g, 10 mmol) were dissolved in 12.5mL methanol. One drop of acetic acid was then added, followed by 1.55 g2,2-dimethyl-heptan-3-one. The mixture was stirred at room temperaturefor several days, at which time 2.1 mL acetic acid and 667 mg NaBH₃CNwere added. After stirring for ca. 7 hours, the methanol was removed invacuo. The residual product was diluted with ca. 20 mL of water andextracted with methylene chloride. The extracts were dried over MgSO₄,filtered from solids, and solvent was removed in vacuo to provide 1.8 gcrude product. This material was purified by column chromatography onsilica gel, eluting with a 100% hexanes −100% ethyl ether gradient.4-Ethyl-benzoic acid N′-(1-tert-butyl-pentyl)-hydrazide was recovered in45% yield (1.32 g).

4-Ethyl-benzoic acid N′-(1-tert-butyl-pentyl)-hydrazide (145.2 mg, 0.5mmol) was dissolved in 5 mL methylene chloride and 1.5 mmol PS-NMM (804mg, a —SO₂NH(CH₂)₃-morpholine functionalized polystyrene resin availablefrom Argonaut Technologies, San Carlos, Calif.) was added. The mixturewas diluted with 3 ml methylene chloride to generate a stirrablesuspension. 3,5-dimethylbenzoyl chloride (0.5 mmol, 74 mL) was added andthe mixture was stirred overnight. The following day, 1 mmol (775 mg) ofAP-NCO resin (isocyanate-functionalized resin available from ArgonautTechnologies, San Carlos, Calif.) and 1 mmol (401.6 mg) of AP-trisamine(polystyrene-CH₂NHCH₂CH₂NH(CH₂CH₂NH₂)₂ resin available from ArgonautTechnologies, San Carlos, Calif.) were added with 3 mL methylenechloride to scavenge remaining starting material. The mixture wasstirred for 4 hours, the resins were filtered away, and the filtrate wasdried to provide 191 mg crude product which indicated one spot by TLCanalysis. This material was purified by flash chromatography on silicagel using a gradient of 100% hexane-100% ethyl ether. Yield: 50 mg (ca.23%) 3,5-dimethyl-benzoic acidN-(1-tert-butyl-pentyl)-N′-(4-ethyl-benzoyl)-hydrazide. ¹H NMR (CDCl₃,400 MHz) δ (ppm): 7.8+7.5 (br/br, 1H), 7.4-6.9 (m, 7H), 4.7+3.6 (m/m,1H), 2.65 (m, 2H), 2.38+2.28 (s/s, 6H), 1.9+1.75 (br, 2H), 1.4-1.2 (br,m, 7H), 1.1 (br s, 9H), 0.95 (br s, 3H).

Reporter Assays: Cells were harvested 40 hours after adding ligands. 125μL of passive lysis buffer (part of Dual-luciferase™ reporter assaysystem from Promega Corporation) were added to each well of the 24-wellplate. The plates were placed on a rotary shaker for 15 minutes. TwentyμL of lysate were assayed. Luciferase activity was measured usingDual-luciferase reporter assay system from Promega Corporation followingthe manufacturer's instructions. Fold induction (FI) activities werecalculated by dividing relative light units (“RLU”) in ligand treatedcells with RLU in DMSO treated cells (untreated control).

Example 3

This Example describes the identification of CfEcR ligand binding domainsubstitution mutants that are generally ecdysteroid responsive thatexhibit increased activity in response to ecdysteroids. In an effort toidentify substitution mutations in the CfEcR that increase ecdysteroidactivity, Applicants mutated amino acid residues and createdGAL4/mutantCfEcR-DEF cDNA gene expression cassettes as described inExample 1 above using PCR-mediated site-directed mutagenesis kit. Themutated and the WT cDNAs corresponding to the various switch constructsoutlined above in Example 1.1 and 1.2 were made and tested in aGAL4-driven luciferase reporter assay as described in Example 2.

Specific amino acid residues were identified that, when substituted,yield a mutant ecdysone receptor that exhibits increased activity inresponse to an ecdysteroid ligand. The effect of an amino acidsubstitution at amino acid residue 119 of SEQ ID NO: 1 on the activityof the mutated CfEcR-DEF receptor is presented in Table 3a as a foldincrease over Gal4/wild-type CfEcR-DEF (WT) switch activity. The effectof an amino acid substitution at amino acid residue 96 of SEQ ID NO: 1and double amino acid substitution at amino residues 96 and 119 on theactivity of the mutated CfEcR-DEF receptor is presented in Table 3b asEC₅₀ and relative maximum fold induction. EC₅₀s were calculated fromdose response data using a three-parameter logistic model. Relative MaxFI was determined as the maximum fold induction of the tested ligand (anembodiment of the invention) observed at any concentration relative tothe maximum fold induction of GS®-E ligand (RG-102240;3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide) observed at anyconcentration.

TABLE 3a CfEcR-DEF mutant that shows increased ecdysteroid activity Foldincrease over WT N119F 1.6 nM GS ®-E ligand (RG-102240) 1.22 8 nM GS ®-Eligand (RG-102240) 0.73 40 nM GS ®-E ligand (RG-102240) 0.06 200 nMGS ®-E ligand (RG-102240) 0.01 1 μM GS ®-E ligand (RG-102240) 0.08 5 μMGS ®-E ligand (RG-102240) 0.59 1.6 nM PonA 1.33 8 nM PonA 1.7 40 nM PonA9.42 200 nM PonA 6.50 1 μM PonA 3.00

TABLE 3b CfEcR-DEF mutants that shows increased ecdysteroid activity DAHTHQ THQ DAH RG- DAH RG- RG- RG- RG- ECD ECD Mutant 102240 101691 102362120499 120500 20E PonA V96S EC50 1.14 0.87 2.07 >33 >33 >33 ~2 (μM) V96SRel 1 0.9 0.57 0 0 0.02 0.92 Max FI N119F/V96T EC50 ~8 3.63 ~10 ~20 >33~8 ~0.3 (μM) N119F/V96T Rel 1 0.13 0.19 0.1 0.02 0.46 2.02 Max FI

As seen in Tables 3a and 3b, the activity of ecdysteroids was increasedsignificantly when the CfEcR ligand binding domain was mutated at aminoacid residues 96 or 119 of SEQ ID NO: 1 and double mutated at amino acidresidues 96 and 119 of SEQ ID NO: 1, indicating that these residues areimportant residues in the ligand binding pocket of CfEcR.

Example 4

This Example describes the identification of additional CfEcR ligandbinding domain substitution mutants that are generally non-ecdysteroiddiacylhydrazine responsive that exhibit increased activity in responseto diacylhydrazine ligands. In an effort to identify substitutionmutations in the CfEcR that increase diacylhydrazine ligand activityApplicants mutated amino acid residues predicted to be critical forecdysteroid binding and created GAL4/mutantCfEcR-DEF cDNA geneexpression cassettes as described in Example 1 above using PCR-mediatedsite-directed mutagenesis kit. The mutated and the WT cDNAscorresponding to the various switch constructs outlined above in Example1.1 and 1.2 were made and tested in GAL4-driven luciferase reporterassays as described in Example 2.

Specific amino acid residues were identified that, when substituted,yield mutant ecdysone receptors that exhibit increased activity inresponse to non-ecdysteroid diacylhydrazine ligands. The effect of anamino acid substitution at amino acid residue 48, 52, 54, 109, 110, 125,132 and 223 of SEQ ID NO: 1 and a double substitution at amino acidresidues 52 and 110 of SEQ ID NO: 1 on the activity of the mutatedCfEcR-DEF receptor is presented in Tables 4a and 4b as EC₅₀ and relativemaximum fold induction. EC₅₀s were calculated from dose response datausing a three-parameter logistic model. Relative Max FI was determinedas the maximum fold induction of the tested ligand (an embodiment of theinvention) observed at any concentration relative to the maximum foldinduction of GS®-E ligand (3,5-dimethyl-benzoic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide) observed at anyconcentration.

TABLE 4a CfEcR mutants that show increased diacylhydrazine ligandactivity DAH RG- DAH DAH THQ THQ ECD ECD Mutant 102240 RG-101691RG-102362 RG-120499 RG-120500 20E PonA A110E EC50 (μM) 0.59 0.851.02 >33 >33 >33 ~10 A110E Rel Max FI 1 1.03 0.78 0 0.01 0 1.09 A110NEC50 (μM) 1.41 0.88 0.72 >33 >33 >33 >33 A110N Rel Max FI 1 0.99 0.86 00 0 0 F109M EC50 (μM) 0.66 0.65 0.82 >33 ~20 >33 ~20 F109M Rel Max FI 10.75 0.6 0.04 0.05 0 0.16 A110P EC50 (μM) 0.55 0.67 0.77 >33 >33 >33 >33A110P Rel Max FI 1 0.89 0.64 0 0 0 0 F48Y EC50 (μM) 1.27 0.930.59 >33 >33 >33 ~10 F48Y Rel Max FI 1 0.69 0.48 0 0 0 0.33 F48W EC50(μM) ~1 1.53 0.77 >33 >33 >33 ~10 F48W Rel Max FI 1 0.74 0.51 0 0 0 0.65F48L EC50 (μM) 0.46 0.3 0.56 >33 >33 >33 ~10 F48L Rel Max FI 1 0.81 0.530 0 0 0.59 M54T EC50 (μM) 0.08 0.03 0.05 >33 >33 >33 9.46 M54T Rel MaxFI 1 0.71 0.66 0 0 0 0.5 T52L EC50 (μM) ~0.5 0.21 0.33 >33 >33 >33 5.03T52L Rel Max FI 1 0.74 0.61 0 0 0 0.54 T52V/A110P EC50 (μM) 0.33 0.240.32 >33 >33 >33 >33 T52V/A110P Rel Max FI 1 0.66 0.94 0 0 0 0

TABLE 4b CfEcR mutants that show increased diacylhydrazine ligandactivity Mutant RG-102240 RG-115840 RG-115853 RG-115855 RG-115859RG-115898 F48R EC50 7.23 3.58 0.1 5.18 9.41 >33 (μM) F48R Rel Max 1 2.655.71 1.02 1.25 0 FI L132E EC50 0.41 1.67 1.54 0.45 0.08 2.15 (μM) L132ERel Max 1 1.56 1.12 1.15 0.51 0.34 FI M125I EC50 1.36 3.02 1.53 20.28 >33 (μM) M125I Rel Max 1 0.45 0.54 0.69 0.76 0 FI L223Y EC50 3.150.58 0.65 0.3 1.27 0.33 (μM) L223Y Rel Max 1 1.42 0.77 1.6 0.79 0.2 FIM125G EC50 14.17 2.97 0.14 0.08 5 0.08 (μM) M125G Rel Max 1 47.96 39.4146.54 3.14 28.81 FI M125N EC50 9.88 3.3 0.5 0. > 33 8 0.94 (μM) M125NRel Max 1 22.56 11.64 25.3 4.11 11.57 FI

As seen in Tables 4a and 4b, the activity of diacylhydrazines wasincreased significantly when the CfEcR ligand binding domain was mutatedat amino acid residues 48, 52, 54, 109, 110, 125, 132 and 223 of SEQ IDNO: 1 and double mutated at amino acid residues 52 and 110 of SEQ ID NO:1, indicating that these residues are important residues in the ligandbinding pocket of CfEcR.

Example 5

This Example describes the identification of additional CfEcR ligandbinding domain substitution mutants that are generally diacylhydrazineand ecdysteroid responsive that exhibit increased activity in responseto diacylhydrazine ligand and ecdysteroid. In an effort to identifysubstitution mutations in the CfEcR that increase diacylhydrazine ligandactivity and ecdysteroid ligand activity, Applicants mutated amino acidresidues and created GAL4/mutantCfEcR-DEF cDNA gene expression cassettesas described in Example 1 above using PCR-mediated site-directedmutagenesis kit. The mutated and the WT cDNAs corresponding to thevarious switch constructs outlined above in Example 1.1 and 1.2 weremade and tested in GAL4-driven luciferase reporter assays as describedin Example 2. The effect of an amino acid substitution at amino acidresidue 109, 132, 238 of SEQ ID NO: 1 or substitution at amino acidresidues 52, 107 and 127 of SEQ ID NO: 1 or 107, 127 and addition of aglycine at the end of SEQ ID NO: 1 on the activity of the mutatedCfEcR-DEF receptor is presented in Table 5.

TABLE 5 CfEcR mutants that show increased diacylhydrazine andecdysteroid activity DAH DAH DAH THQ THQ RG- RG- RG- RG- RG- ECD ECDMutant 102240 101691 102362 120499 120500 20E PonA F109W EC50 0.61 0.491.41 >33 >33 >33 4.06 (μM) F109W Rel Max 1 0.79 0.7 0.01 0.01 0 0.08 FIV107I/Y127E/T52V EC50 0.06 0.02 <0.01 >33 >33 >33 ~1 (μM)V107I/Y127E/T52V Rel Max 1 0.88 0.73 0 0 0.04 0.67 FI V107I/Y127E/G EC500.17 0.02 0.06 >33 >33 >33 1.65 (μM) V107I/Y127E/G Rel Max 1 0.85 0.81 00 0.03 0.67 FI L132M EC50 0.77 0.51 0.13 >33 >33 >33 5.47 (μM) L132M RelMax 1 0.77 0.66 0.01 0.04 0 0.57 FI L132V EC50 2.32 0.660.29 >33 >33 >33 6.56 (μM) L132V Rel Max 1 0.77 0.74 0.01 0.04 0 0.57 FIW238P EC50 ~0.4 0.65 0.29 >33 >33 >33 ~3.3 (μM) W238P Rel Max 1 0.91 0.40.01 0.01 0 0.73 FI

As seen in Table 5, both diacylhyrazine and ecdysteroid activities wereincreased when the CfEcR ligand binding domain was mutated at amino acidresidues 48, 51, 52, 54, 96, 120, 125, 128, 132, 234 and 238, indicatingthat these residues are important residues in the ligand binding pocketof CfEcR.

Example 6

This Example describes the identification of additional CfEcR ligandbinding domain substitution mutants that are generally diacylhydrazineand tetrahydroquinoline responsive that exhibit increased activity inresponse to diacylhydrazine and tetrahydroquinoline ligands. In aneffort to identify substitution mutations in the CfEcR that increasediacylhydrazine ligand activity and tetrahydroquinoline ligand activity,Applicants mutated amino acid residues predicted and createdGAL4/mutantCfEcR-DEF cDNA gene expression cassettes as described inExample 1 using PCR-mediated site-directed mutagenesis kit. The mutatedand the WT cDNAs corresponding to the various switch constructs outlinedabove in Example 1.1 and 1.2 were made and tested in GAL4-drivenluciferase reporter assays as described in Example 2. The effect oftriple mutations at amino acid residues 107, 110 and 127 of SEQ ID NO: 1and double mutations at 107 and 127 of SEQ ID NO: 1 on the activity ofthe mutated CfEcR-DEF receptor is presented in Table 6.

TABLE 6 CfEcR mutants that show increased diacylhydrazine andtetrahydroquinoline activity 1 2 3 4 5 6 7 DAH DAH DAH THQ THQ ECD ECDMutant RG-102240 RG-101691 RG-102362 RG-120499 RG-120500 20E PonAV107I/Y127E/A110P EC50 (μM) 0.30 0.34 0.10 ~20 3.71 >33 >33V107I/Y127E/A110P Rel Max FI 1.00 0.96 0.63 0.08 0.16 0.00 0.01V128F/A110P EC50 (μM) ~8 ~5 0.45 0.28 >33 >33 V128F/A110P Rel Max FI1.00 0.08 0.51 0.68 0.00 0.00

As seen in Table 6, both non-ecdysteroid, diacylhyrazine andtetrahydroquinoline activities were increased when the CfEcR ligandbinding domain was mutated at amino acid residues 107, 110 and 127 and107 and 127, indicating that these residues are important residues inthe ligand binding pocket of CfEcR.

Example 7

Table 7 describes the effect of the diacylhydrazine GS™-E ligand versusthe DMSO control at various concentrations on the maximum fold inductionof various CfEcR mutants.

TABLE 7 Effect of GS ™-E ligand v. DMSO control on the maximum foldinduction of CfEcR mutants. GS ™-E ligand max FI (relative ConcentrationMutant to DMSO) (mM) at max FI A110E 4926 10.00 A110N 1678 10.00 A110W5207 10.00 F109W 3063 10.00 F109P 1 10.00 F109L 20 33.30 F109M 1475 3.3F109N 1506 33.3 F48Y 1355 33.3 F48W 1638 33.3 F48L 2599 33.3 I51N 133.30 I51L 2478 33.30 L132M 1517 10.00 L132N 785 33.30 L132V 2128 10.00L234M 4578 33.30 L234I 2650 10 M125P 1 33.3 M125R 2407 33.3 M125C 9 33.3M54W 1678 10 M54T 4460 10.00 M92L 1203 33.30 M92E 141 33.30 R95H 341333.30 R95M 1691 33.30 R95W 1820 33.30 T52L 1128 33.3 T52E 1537 33.3 V96L4378 10 V96W 615 33.3 V96S 1828 33.3 W238P 4812 10 W238E 1018 33.3 W238Y11 33.3 Y120W 1889 33.3 Y120M 1708 33.3 N119F/V96T 1738 33.3 V107I/Y127E3146 10 V107I/Y127E/A110P 2212 10 M125E 1196 33.3 M125L 2250 33.3 T52P301 33.3 V96E 2963 33.30 A110P 2289 3.30 V128F/A110P 2960 33.30 V128F550 33.30

Example 8

This Example describes the identification of additional CfEcR ligandbinding domain substitution mutants that exhibit decreased activity inresponse to diacylhydrazine ligands. In an effort to identifysubstitution mutations in the CfEcR that decrease diacylhydrazine ligandactivity, Applicants mutated amino acid residues predicted to becritical in diacylhydrazine binding and created GAL4/mutantCfFcR-DEFcDNA gene expression cassettes as described in Example 1 usingPCR-mediated site-directed mutagenesis kit. The mutated and the WT cDNAscorresponding to the various switch constructs outlined above in Example1.1 and 1.2 were made and tested in GAL4-driven luciferase reporterassays as described in Example 2. The effect of an amino acidsubstitution at amino acid residue 48, 51, 52, 54, 92, 95, 96, 109, 120,125, 219, 223, 234 or 238 of SEQ ID NO: 1 on the activity of the mutatedCfEcR-DEF receptor is presented in Tables 8a and 8b.

TABLE 8a CfEcR mutants that show decreased diacylhydrazine activity DAHTHQ THQ DAH RG- DAH RG- RG- RG- RG- ECD ECD Mutant 102240 101691 102362120499 120500 20E PonA M92L EC50 ~8 9.9 ~20 >33 >33 >33 >33 (μM) M92LRel Max 1 0.41 0.05 0 0 0 0 FI M92E EC50 ~8 ~20 >33 >33 >33 >33 >33 (μM)M92E Rel Max 1 0.08 0.04 0 0 0 0.01 FI R95W EC50 ~7 ~10~8 >33 >33 >33 >33 (μM) R95W Rel Max 1 0.37 0.25 0 0 0 0 FI T52E EC50 ~7~7 7.16 >33 >33 >33 >33 (μM) T52E Rel Max 1 0.57 0.34 0 0 0 0 FI W238EEC50 ~6 ~8 3.45 >33 >33 >33 >33 (μM) W238E Rel Max 1 0.71 0.45 0 0 0 0FI Y120M EC50 ~4 ~10 ~10 >33 >33 >33 >33 (μM) Y120M Rel Max 1 0.3 0.13 00 0 0.04 FI I51L EC50 3.2 2.28 3.35 >33 >33 >33 ~8 (μM) I51L Rel Max 10.88 0.53 0 0 0 0.66 FI V96W EC50 ~1 3.61 3.26 >33 >33 >33 ~3.3 (μM)V96W Rel Max 1 0.75 0.38 0 0.01 0 0.83 FI Y120W EC50 4.21 9.764.96 >33 >33 >33 ~10 (μM) Y120W Rel Max 1 0.89 0.67 0.02 0.01 0 0.69 FIW238Y EC50 ~13 >33 >33 >33 >33 >33 >33 (μM) W238Y Rel Max 1 0.3 0.060.13 0.1 0.18 0.07 FI F109N EC50 2.7 3.95 1.85 >33 >33 >33 >33 (μM)F109N Rel Max 1 0.85 0.4 0 0 0 0 FI L234M EC50 1.43 1.792.04 >33 >33 >33 >33 (μM) L234M Rel Max 1 0.77 0.43 0 0 0 0.02 FI M125EEC50 ~2 0.98 0.83 >33 >33 >33 >33 (μM) M125E Rel Max 1 0.53 0.4 0 0 0 0FI V96E EC50 ~2 1.62 1.86 >33 >33 >33 >33 (μM) V96E Rel Max 1 0.81 0.480 0 0 0.02 FI F48N EC50 0.75 1.73 1.68 >33 >33 >33 ~20 (μM) F48N Rel Max1 0.88 0.66 0 0 0 0.17 FI L234I EC50 0.77 0.94 2.46 >33 >33 >33 ~7 (μM)L234I Rel Max 1 0.73 0.44 0 0.01 0 0.56 FI M54W EC50 1.17 1.631.24 >33 >33 >33 ~10 (μM) M54W Rel Max 1 0.75 0.44 0.01 0.01 0 0.46 FIV96L EC50 ~1 1.68 2.67 >33 >33 >33 7.49 (μM) V96L Rel Max 1 0.62 0.58 00 0 0.49 FI

TABLE 8b CfEcR mutants that show decreased diacylhydrazine activityMutant RG-102240 RG-115840 RG-115853 RG-115855 RG-115859 RG-115898 I51MEC50 3.94 4.13 2.94 1.33 2 >33 (μM) Rel Max 1 0.27 0.46 0.84 1.07 0.02FI L234R EC50 17.1 20 >33 >33 20 >33 (μM) Rel Max 1 2.24 0.2 0.21 3.80.58 FI L234W EC50 11.48 >33 >33 6 5 >33 (μM) Rel Max 1 0.06 0.07 0.440.42 0.02 FI M219A EC50 2.9 2.87 3.65 1.44 3.18 1 (μM) Rel Max 1 0.60.79 0.86 0.85 0.05 FI L223K EC50 3.93 1 2.5 1.23 0.38 0.28 (μM) Rel Max1 0.93 0.54 0.87 0.9 0.18 FI M125V EC50 1.64 3.79 1.72 2 1 >33 (μM) RelMax 1 0.47 0.5 0.74 0.87 0.01 FI M219K EC50 2.9 >33 3.31 1.93 5 >33 (μM)Rel Max 1 0.01 0.22 0.34 0.66 0 FI M219W EC50 3.35 2.33 ~20 2 4 >33 (μM)Rel Max 1 0.39 0.12 0.34 0.46 0 FI M219Y EC50 0.82 1 ~20 2 2 >33 (μM)Rel Max 1 0.68 0.05 0.32 0.51 0.01 FI T52M EC50 6.74 5 1.36 10 3.56 >33(μM) Rel Max 1 0.15 0.32 0.66 1.08 0.01 FI T52R EC50 6.69 >33 5 3.316.14 >33 (μM) Rel Max 1 0.02 0.06 0.1 0.41 0 FI W238L EC50 11.13 2 >33 25 >33 (μM) Rel Max 1 0.41 0.02 0.09 1.08 0 FI W238M EC50 10.47 2 >33 21.85 >33 (μM) Rel Max 1 0.41 0.05 0.72 16.07 0.03 FI F48K EC50 11.093.76 4.42 2.45 >33 >33 (μM) Rel Max 1 0.18 0.71 4.78 0.02 0.08 FI T52GEC50 3.52 3.61 3.35 2 6.49 >33 (μM) Rel Max 1 0.23 0.28 0.31 0.44 0.01FI T52Q EC50 2.95 2 >33 0.56 1.11 20 (μM) Rel Max 1 0.34 0.04 0.39 1.020.08 FI L223R EC50 8.69 2 2 1.2 5.19 >33 (μM) Rel Max 1 0.25 0.09 1.350.61 0.01 FI

As seen in Tables 8a and 8b, the activity of diacylhydrazines wasdecreased significantly when the CfEcR ligand binding domain was mutatedat amino acid residues 48, 51, 52, 54, 92, 95, 96, 109, 120, 125, 219,223, 234 or 238 of SEQ ID NO: 1, indicating that these residues areimportant residues in the ligand binding pocket of CfEcR.

Example 9

This Example describes the identification of additional CfEcR ligandbinding domain substitution mutants that are generallytetrahydroquinoline responsive that exhibit increased activity inresponse to tetrahydraquinoline ligands. In an effort to identifysubstitution mutations in the CfEcR that increase tetrahydroquinolineligand activity, Applicants mutated specific amino acid residues andcreated GAL4/mutantCfEcR-DEF cDNA gene expression cassettes as describedin Example 1 using PCR-mediated site-directed mutagenesis kit. Themutated and the WT cDNAs corresponding to the various switch constructsoutlined above in Example 1.1 and 1.2 were made and tested inGAL4-driven luciferase reporter assays as described in Example 2. Theeffect of an amino acid substitution at amino acid residue 110 or 128 ofSEQ ID NO: 1 or the double amino acid substitution at amino acidresidues 110 and 128 of SEQ ID NO: 1 on the activity of the mutatedCfEcR-DEF receptor is presented in Table 9.

TABLE 9 CfEcR mutants that show increased tetrahydroquinoline activityDAH DAH DAH THQ THQ RG- RG- RG- RG- RG- ECD ECD Mutant 102240 101691102362 120499 120500 20E PonA A110W EC50 1.37 1.06 2.99 ~10 ~5 >33 >33(μM) A110W Rel Max 1 0.8 0.55 0.06 0.07 0 0.01 FI V128F EC50 >33 ~8.3,5.4 >33 >33 ~10 ~10 >33 (μM) V128F Rel Max 0 0.04 2.34 0.05 1 0.02 0.03FI V128F/A110P EC50 ~8 ~5 0.45 0.28 >33 >33 (μM) V128F/A110P Rel Max 10.08 0.51 0.68 0 0 FI

As seen in Table 9, the activity of tetrahydroquinolines was increasedsignificantly when the CfEcR ligand binding domain was mutated at aminoacid residues 110 or 128 of SEQ ID NO: 1 or doubled mutated at aminoacid residues 110 and 128 of SEQ ID NO: 1, indicating that theseresidues are important residues in the ligand binding pocket of CfEcR.

Example 10

This Example describes the identification of additional CfEcR ligandbinding domain substitution mutants that are differentially responsiveto diacylhydrazine ligands. These mutants exhibit a general decrease indiacylhydrazine activity; however they are selectively responsive to aspecific diacylhydrazine ligand. In an effort to identify substitutionmutations in the CfEcR, Applicants mutated specific amino acid residuesand created GAL4/mutantCfEcR-DEF cDNA gene expression cassettes asdescribed in Example 1 using PCR-mediated site-directed mutagenesis kit.The mutated and the WT cDNAs corresponding to the various switchconstructs outlined above in Example 1.1 and 1.2 were made and tested inGAL4-driven luciferase reporter assays as described in Example 2. Theeffect of an amino acid substitution at amino acid residue 52, 95, 109,125 or 132 of SEQ ID NO: 1 on the activity of the mutated CfEcR DEFreceptor is presented in Tables 10a and 10b.

TABLE 10a CfEcR mutants that show decreased diacylhydrazine activity andincreased activity in response to diacylhydrazine RG-115855 RG- RG- RG-RG- RG- RG- Mutant 102240 101691 102362 115855 120499 120500 20E PonAF109L EC50 ~2 2.34 ~2.5 1.73 >33 >33 >33 >33 (μM) F109L Rel Max 1 0.680.17 1.01 0.03 0.02 0.09 0.03 FI L132M EC50 ~12 ~20 >330.39 >33 >33 >33 >33 (μM) L132M Rel Max 1 0.08 0.01 0.90 0 0 0.01 0.01FI R95H EC50 1.58 3.9 3.49 0.78 >33 >33 >33 >33 (μM) R95H Rel Max 1 0.960.62 0.68 0 0 0 0.03 FI R95M EC50 2.76 3.3 4.28 3.74 >33 >33 >33 >33(μM) R95M Rel Max 1 0.57 0.27 0.33 0 0.01 0 0 FI M125L EC50 ~10 >33 >330.16 >33 >33 >33 >33 (μM) M125L Rel Max 1 0 0.01 2.15 0 0 0 0 FI T52PEC50 ~10 >33 ~6 3.87 >33 >33 >33 >33 (μM) T52P Rel Max 1 0.02 0.11 1.930.03 0.03 0.02 0.03 FI M125W EC50 3.49 4.94 3.5 0.03 >33 >33 >33 >33(μM) M125W Rel Max 1 0.74 0.44 1.24 0 0 0 0 FI M125R EC50 3.7 ~10 10.380.02 >33 >33 >33 ~8 (μM) M125R Rel Max 0 0.01 0 1.39 0 0.01 0 FI M125CEC50 ~8, >33 33 33 0.58 33 33 33 33 (μM) M125C Rel Max 1 0.62 0.15876.58 0.27 0.14 0.22 0.28 FI M125P EC50 >33 >33 >330.45 >33 >33 >33 >33 (μM) M125P Rel Max 1 5.25 0.78 380.86 0.65 1.3 1.290.7 FI

TABLE 10b CfEcR mutants that show decreased RG-102240 diacylhydrazineactivity and increased activity in response to other diacylhydrazinesMutant RG-102240 RG-115840 RG-115853 RG-115855 RG-115859 RG-115898 M125SEC50 12.33 1.4 0.98 0.11 7.26 0.33 (μM) Rel Max 1 22.73 15.97 25.22 6.3916.98 FI T52W EC50 18.33 4.07 2 0.96 7 0.18 (μM) Rel Max 1 30.59 89.3249.21 2.81 4.24 FI

As seen in Tables 10a and 10b, the activity of diacylhydrazines wasdifferentially affected when the CfEcR ligand binding domain was mutatedat amino acid residues 52, 95, 109, 125 or 132 of SEQ ID NO: 1,indicating that these residues are important residues in the ligandbinding pocket of CfEcR.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

1. A gene expression modulation system comprising a gene expressioncassette that is capable of being expressed in a host cell, the geneexpression cassette comprising a polynucleotide that encodes apolypeptide comprising: i) a transactivation domain; ii) a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and iii) a Group H nuclear receptorligand binding domain comprising at least one mutation, wherein themutation is at a position equivalent to or analogous to amino acidresidues selected from the group consisting of: a) at least one of 48,51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223,234, and 238 of SEQ ID NO: 1, b) both 96 and 119 of SEQ ID NO: 1, c)both 110 and 128 of SEQ ID NO: 1, d) both 52 and 110 of SEQ ID NO: 1, e)both 107, 110, and 127 of SEQ ID NO: 1, f) both 52, 107 and 127 of SEQID NO: 1 and g) both 107, 127 and 259 of SEQ ID NO: 1, or anycombination thereof.
 2. The gene expression modulation system accordingto claim 1, further comprising a second nuclear receptor ligand bindingdomain selected from the group consisting of a vertebrate retinoid Xreceptor ligand binding domain, an invertebrate retinoid X receptorligand binding domain, an ultraspiracle protein ligand binding domain,and a chimeric ligand binding domain comprising two polypeptidefragments, wherein the first polypeptide fragment is from a vertebrateretinoid X receptor ligand binding domain, an invertebrate retinoid Xreceptor ligand binding domain, or an ultraspiracle protein ligandbinding domain, and the second polypeptide fragment is from a differentvertebrate retinoid X receptor ligand binding domain, invertebrateretinoid X receptor ligand binding domain, or ultraspiracle proteinligand binding domain.
 3. A gene expression modulation systemcomprising: a) a first gene expression cassette that is capable of beingexpressed in a host cell comprising a polynucleotide that encodes afirst polypeptide comprising: i) a DNA-binding domain that recognizes aresponse element associated with a gene whose expression is to bemodulated; and ii) a nuclear receptor ligand binding domain; and b) asecond gene expression cassette that is capable of being expressed inthe host cell comprising a polynucleotide that encodes a secondpolypeptide comprising: i) a transactivation domain; and ii) a nuclearreceptor ligand binding domain wherein one of the nuclear receptorligand binding domains is a Group H nuclear receptor ligand bindingdomain comprising at least one mutation, wherein the mutation is at aposition equivalent to or analogous to amino acid residues selected fromthe group consisting of: a) at least one of 48, 51, 52, 54, 92, 95, 96,109, 110, 119, 120, 125, 128, 132, 219, 223, 234 and 238 of SEQ ID NO:1, b) both 96 and 119 of SEQ ID NO: 1, c) both 110 and 128 of SEQ ID NO:1, d) both 52 and 110 of SEQ ID NO: 1, e) all three of 107, 110, and 127of SEQ ID NO: 1, f) all three of 52, 107 and 127 of SEQ ID NO: 1 and g)all three of 107, 127 and 259 of SEQ ID NO: 1, or any combinationthereof.
 4. The gene expression modulation system according to claim 1or claim 3, wherein the Group H nuclear receptor ligand binding domainis from a Group H nuclear receptor selected from the group consisting ofan ecdysone receptor, a ubiquitous receptor, an orphan receptor 1, aNER-1, a steroid hormone nuclear receptor 1, a retinoid X receptorinteracting protein-15, a liver X receptor β, a steroid hormone receptorlike protein, a liver X receptor, a liver X receptor α, a farnesoid Xreceptor, a receptor interacting protein 14, and a farnesol receptor. 5.The gene expression modulation system according to claim 4, wherein theGroup H nuclear receptor ligand binding domain is encoded by apolynucleotide comprising at least one mutation that results in at leastone mutation of an amino acid residue, wherein the amino acid residue isat a position equivalent to or analogous to: a) at least one of aminoacid residue 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128,132, 219, 223, 234, and 238 of SEQ ID NO: 1, b) both amino acid residues96 and 119 of SEQ ID NO: 1, c) both amino acid residues 110 and 128 ofSEQ ID NO: 1, d) both amino acid residues 52 and 110 of SEQ ID NO: 1, e)all three amino acid residues 107, 110, and 127 of SEQ ID NO: 1, f) allthree amino acid residues 52, 107 and 127 of SEQ ID NO: 1 or g) allthree amino acid residues 107, 127 and 259 of SEQ ID NO: 1, or anycombination thereof.
 6. The gene expression modulation system accordingto claim 5, wherein the Group H nuclear receptor ligand binding domaincomprising a substitution mutation is an ecdysone receptor ligandbinding domain.
 7. The gene expression modulation system according toclaim 6, wherein the mutation is selected from the group consisting ofF48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, I51L, T52M, T52V, T52L,T52E, T52P, T52R, T52W, T52G, T52Q, M54W, M54T, M92L, M92E, R95H, R95M,R95W, V96L, V96W, V96S, V96E, F109W, F109P, F109L, F109M, F109N, A110E,A110N, A110W, N119F, Y120W, Y120M, M125P, M125R, M125E, M125L, M125C,M125W, M125G, M1251, M125N, M125S, M125V, V128F, L132M, L132N, L132V,L132E, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L2341,L234R, L234W, W238P, W238E, W238Y, W238M, W238L, N119F/V96T,V128F/A110P, T52V/A110P, V107V/Y127E/T52V, V107I/Y127E/G259 andV1071/Y127E/A110P of SEQ ID NO:
 1. 8. The gene expression modulationsystem according to claim 1 or claim 3, wherein the DNA-binding domainis selected from the group consisting of an ecdysone receptorDNA-binding domain, a GAL4 DNA-binding domain, and a LexA DNA-bindingdomain.
 9. The gene expression modulation system according to claim 1 orclaim 3, wherein the transactivation domain is selected from the groupconsisting of an ecdysone receptor transactivation domain, a VP16transactivation domain, a B42 acidic activator transactivation domain,and a p65 transactivation domain.
 10. An isolated polynucleotideencoding a Group H nuclear receptor ligand binding domain comprising atleast one mutation, wherein the isolated polynucleotide comprises atleast one mutation that results in at least one mutation of an aminoacid residue at a position equivalent to or analogous to a) at least oneof amino acid residue 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120,125, 128, 132, 219, 223, 234, and 238 of SEQ ID NO: 1, b) both aminoacid residues 96 and 119 of SEQ ID NO: 1, c) both amino acid residues110 and 128 of SEQ ID NO: 1, d) both amino acid residues 52 and 110 ofSEQ ID NO: 1, e) all three amino acid residues 107, 110, and 127 of SEQID NO: 1, f) all three amino acid residues 52, 107 and 127 of SEQ ID NO:1 or g) all three amino acid residues 107, 127 and 259 of SEQ ID NO: 1or any combination thereof.
 11. The isolated polynucleotide according toclaim 10, wherein the mutation results in a mutation selected from thegroup consisting of F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N,I51L, T52M, T52V, T52L, T52E, T52P, T52R, T52W, T52G, T52Q, M54W, M54T,M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, F109W, F109P,F109L, F109M, F109N, A110E, A110N, A110W, NI 19F, Y120W, Y120M, M125P,M125R, M125E, M125L, M125C, M125W, M125G, M1251, M125N, M125S, M125V,V128F, L132M, L132N, L132V, L132E, M219K, M219W, M219Y, M219A, L223K,L223R, L223Y, L234M, L234I, L234R, L234W, W238P, W238E, W238Y, W238M,W238L, N119F/V96T, V128F/A110P, T52V/A110P, V107I/Y127E/T52V,V1071/Y127E/G259 and V107I/Y127E/A110P of SEQ ID NO:
 1. 12. Anexpression vector comprising the isolated polynucleotide of claim 10operatively linked to a transcription regulatory element.
 13. Anisolated host cell comprising the expression vector of claim 12, whereinthe transcription regulatory element is operative in the host cell. 14.An isolated polypeptide encoded by the isolated polynucleotide accordingto claim
 10. 15. An isolated polypeptide comprising a Group H nuclearreceptor ligand binding domain comprising at least one mutation, whereinthe mutation is at a position equivalent to or analogous to a) at leastone of amino acid residue 48, 51, 52, 54, 92, 95, 96, 109, 110, 119,120, 125, 128, 132, 219, 223, 234, and 238 of SEQ ID NO: 1, b) bothamino acid residues 96 and 119 of SEQ ID NO: 1, c) both amino acidresidues 110 and 128 of SEQ ID NO: 1, d) both amino acid residues 52 and110 of SEQ ID NO: 1, e) all three amino acid residues 107, 110, and 127of SEQ ID NO: 1, f) all three amino acid residues 52, 107 and 127 of SEQID NO: 1 or g) all three amino acid residues 107, 127 and 259 of SEQ IDNO: 1, or any combination thereof.
 16. The isolated polypeptideaccording to claim 15, wherein the mutation is selected from the groupconsisting of F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, I51L,T52M, T52V, T52L, T52E, T52P, T52R, T52W, T52G, T52Q, M54W, M54T, M92L,M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, F109W, F109P, F109L,F109M, F109N, A110E, A110N, A110W, N119F, Y120W, Y120M, M125P, M125R,M125E, M125L, M125C, M125W, M125G, M125I, M125N, M125S, M125V, V128F,L132M, L132N, L132V, L132E, M219K, M219W, M219Y, M219A, L223K, L223R,L223Y, L234M, L234I, L234R, L234W, W238P, W238E, W238Y, W238M, W238L,N119F/V96T, V128F/A110P, T52V/A110P, V107I/Y127E/T52V, V1071/Y127E/G259and V1071/Y127E/A110P of SEQ ID NO:
 1. 17. A method of modulating theexpression of a gene in a host cell comprising the steps of: a)introducing into the host cell the gene expression modulation systemaccording to claim 1 or claim 3; and b) introducing into the host cell aligand; wherein the gene to be modulated is a component of a geneexpression cassette comprising: i) a response element recognized by theDNA binding domain; ii) a promoter that is activated by thetransactivation domain; and iii) a gene whose expression is to bemodulated; whereby upon introduction of the ligand into the host cell,expression of the gene of b)iii) is modulated.
 18. The method accordingto claim 17, wherein the ligand is selected from the group consistingof: a) a compound of the formula:

wherein: E is a branched (C₄-C₁₂)alkyl or branched (C₄-C₁₂)alkenylcontaining a tertiary carbon or a cyano(C₃-C₁₂)alkyl containing atertiary carbon; R¹ is H, Me, Et, i-Pr, F, formyl, CF₃, CHF₂, CHCl₂,CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C^(o)CH, 1-propynyl, 2-propynyl,vinyl, OH, OMe, OEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido, SCN, orSCHF₂; R² is H, Me, Et, n-Pr, i-Pr, formyl, CF₃, CHF₂, CHCl₂, CH₂F,CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C^(o)CH, 1-propynyl, 2-propynyl, vinyl,Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe₂, NEt₂, SMe, SEt, SOCF₃,OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido, OCF₃, OCHF₂,O-i-Pr, SCN, SCHF₂, SOMe, NH—CN, or joined with R³ and the phenylcarbons to which R² and R³ are attached to form an ethylenedioxy, adihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or adihydropyryl ring with the oxygen adjacent to a phenyl carbon; R³ is H,Et, or joined with R² and the phenyl carbons to which R² and R³ areattached to form an ethylenedioxy, a dihydrofuryl ring with the oxygenadjacent to a phenyl carbon, or a dihydropyryl ring with the oxygenadjacent to a phenyl carbon; R⁴, R⁵, and R⁶ are independently H, Me, Et,F, Cl, Br, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CN, C^(o)CH,1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or Set; b) a compound ofthe formula:

wherein: R1 is CH₂CH₃, CH₃, or CH₃; R2 is OCH₃, CH₂CH₃ or i-Pr; and R3and R4 are CH₃; c) a compound of the formula:

wherein: R1 and R2 are F; and R3 is 3-F-4-CH₃-Ph or 3-CH₃-4—F-Ph; and d)an ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, anoxysterol, a 22(R) hydroxycholesterol, 24(S) hydroxycholesterol,25-epoxycholesterol, T0901317,5-alpha-6-alpha-epoxycholesterol-3-sulfate, 7-ketocholesterol-3-sulfate,farnesol, a bile acid, a 1,1-biphosphonate ester, or a Juvenile hormoneIII.
 19. The method according to claim 18, further comprisingintroducing into the host cell a second ligand, wherein the secondligand is 9-cis-retinoic acid or a synthetic analog of a retinoic acid.20. An isolated host cell comprising the gene expression modulationsystem according to claim 1 or claim
 3. 21. A non-human organismcomprising the host cell of claim 20.